Methods and compositions comprising a C-terminal Bax peptide

ABSTRACT

In an aspect, the invention relates to compositions and methods for permeabilizing membranes of cells. In an aspect, the invention relates to compositions and methods for killing cells. In an aspect, the invention relates to compositions and methods of permeabilizing the membranes of cancer cells or microbial cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/717,884,filed May 20, 2015, now U.S. Pat. No. 10,159,706, which is acontinuation of application Ser. No. 14/240,801, filed Feb. 25, 2014,now U.S. Pat. No. 9,040,662. U.S. Pat. No. 9,040,662 is a national stageapplication filed under 35 U.S.C. § 371 of PCT/US2012/052354, filed Aug.24, 2012. PCT/US2012/052354 and U.S. Pat. No. 9,040,662 claim thebenefit of U.S. Provisional Application No. 61/527,524, filed Aug. 25,2011, and U.S. Provisional Application No. 61/645,891, filed May 11,2012. Each of the above listed applications is incorporated herein byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant No. GM083324awarded by the National Institutes of Health. The Government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted May 20, 2015 as a text file named“10613_021US2_2015_05_20_Sequence_Listing.TXT”, created on May 20, 2015,and having a size of 17,889 bytes, is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

The ability to kill cells provides a powerful therapeutic approach totreatment of disease or infection. Bacteria and fungi are becomingresistant to many of the currently available antimicrobial orantimycotic therapeutic compounds. Cancer cells frequently acquiremutations that enhance resistance to standard treatments. Principalamong these mechanisms of drug resistance is abnormal expression ofmembers of the B cell lymphoma-2 (Bcl-2) family (Oltersdorf et al.,2005). The Bcl-2 family consists of more than twenty anti- andpro-apoptotic members that modulate the balance between life and death.Tumors expressing high levels of anti-apoptotic proteins, such as Bcl-2,Mcl -1 or Bcl-xl, can be resistant to the effects of chemotherapeutics(Oltersdorf et al., 2005). This is accomplished, in part, by inhibitionof the pro-apoptotic Bcl-2 family members, such as Bax, first identifiedas a protein that interacts with Bcl-2 (Oltvai et al., 1993). Theassociation of Bax with mitochondria is linked to the release ofcytochrome c and other death-mediators from mitochondrial reserves(Eskes et al., 1998).

Bax is a 21 kD protein of 192 amino acids, comprised of nine alphahelices (Suzuki et al., 2000). Under non-apoptotic conditions, Baxpredominantly resides in the cytosol, with a small percentage of theprotein localized to the mitochondria (Boohaker et al., 2011; Kaufmannet al., 2003; Putcha et al., 1999).

Despite advances in understanding the physiology and pathophysiology ofcancer and/or aberrant cell growth, there is still a scarcity ofcompounds that are efficacious and safe in the treatment of cancerand/or aberrant cell growth. Therapeutics that are effective againstbacteria and fungi are needed as these microorganisms grow moreresistant to current therapies. These needs and other needs aresatisfied by the present invention.

SUMMARY

Disclosed herein is a method of permeabilizing membranes of cells. Suchmembranes may be outer membranes, cell membranes or interior cellularmembranes. The cells may be individual cells or cells in a subject.Methods may comprise administering to at least a cell an effectiveamount of a C-terminal Bax peptide (CT20p peptide) or a compositioncomprising an effective amount of a CT20p peptide.

Disclosed herein is a method of killing cancer cells in a subjectcomprising administering to at least one cell of a subject an effectiveamount of CT20p peptide or a composition comprising an effective amountof CT20p peptide. As used herein, CT20, CT20p peptide or CT20p peptiderefers to a peptide comprising the last 20 amino acids of the BaxC-terminus.

Disclosed herein is a method of killing microbial cells, including butnot limited to bacteria and fungi, whether on a surface, present in acolony or in a subject, comprising administering to at least one cell ofa subject an effective amount of a CT20p peptide or a compositioncomprising an effective amount of a CT20p peptide.

Disclosed herein is a method of permeabilizing membranes of cells. Suchmembranes may be outer membranes, cell membranes or interior cellularmembranes. The cells may be individual cells or cells in a subject. Themethod comprises (i) administering an effective amount of a CT20ppeptide, and (ii) forming at least one pore in a membrane of at leastone cell, wherein the peptide or a composition comprising an effectiveamount of a CT20p peptide destabilizes at least one membrane,facilitates ion exchange, and/or causes a sequestered molecule to bereleased.

Disclosed herein is a method of killing cancer cells in a subjectcomprising (i) administering to a subject an effective amount of a CT20ppeptide, (ii) permeabilizing at least one membrane in a cell of thesubject, and (iii) inducing cell death.

Disclosed herein is a method of killing bacterial or fungal cells in asubject comprising (i) administering to a subject an effective amount ofa CT20p peptide, (ii) permeabilizing at least one membrane in a cell ofthe subject, and (iii) inducing cell death.

Disclosed herein is a composition for permeabilizing membranes on or incells comprising a CT20p peptide.

Disclosed herein is a composition for killing cells comprising a CT20ppeptide.

Disclosed herein is a composition for permeabilizing lipid membranes incells comprising a CT20p peptide and one or more therapeutic agents,including but not limited to antibiotics, antimycotis, and anti-cancerdrugs.

Disclosed herein is a composition for killing cells comprising a CT20ppeptide and one or more therapeutic agents, including but not limited toantibiotics, antimycotis, and anticancer drugs.

DESCRIPTION OF DRAWINGS

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIGS. 1A and 1B show distribution of Bax proteins in lysates. FIG. 1Ashows that the C-terminus of wild-type Bax is distributed amongcytosolic and mitochondria lysates as well as various substitutions atthe C-terminus that modulate the association of the full-length proteinto the mitochondria. FIG. 1B shows additional substitutions thatmodulate the association of the full length protein to the mitochondria.FIG. 1C shows the results of the analysis of the effects of mutations inBax demonstrating that the C-terminus of Bax mediates membranepermeablization.

FIG. 2A shows that most of the DD-tagged Bax full length was found incytosolic extracts. FIGS. 2B and 2C show that DD-tagged CT20 p peptidetranslocated to mitochondria and caused cell death.

FIG. 3A shows a schematic representation of the three dimensionalstructure of aliphatic hyperbranched nanoparticles. FIG. 3B shows aschematic representation of cells taking up nanoparticles. FIG. 3C showsthat DiI-loaded nanoparticles result in minimal to no cell deathcompared to the positive control (dead cells).

FIG. 4A shows changes in cell morphology, including disruption ofmitochondria, cell shrinkage and membrane perturbations that areindicative of cell death of Bax^(+/+) HCT-116 cells. FIG. 4B showschanges in cell morphology, including disruption of mitochondria, cellshrinkage and membrane perturbations that are indicative of cell deathof Bax^(−/−) HCT-116 cells. FIG. 4C shows that cell death was measuredand the loss of membrane integrity was detected within three hours oftreatment with CT20 Bax peptide-nanoparticles.

FIGS. 5A and 5C show morphological changes in MCF-7 (FIG. 5A) andMDS-MB-231 (FIG. 5C) cells following treatment with CT20p peptidenanoparticles. FIGS. 5B and 5D show that the loss of membrane integritywas detected by 3 hours of treatment in MCF-7 (FIG. 5B) and MDS-MB-231(FIG. 5D).

FIGS. 6A and 6B show data for membrane and cellular effects of CT20ppeptide, including that tumor cell death mediated by the CT20p peptideis independent of effector caspases and is resistant to Bcl-2overexpression. FIGS. 6C-D show that CT20p peptide can cause tumorregression in a mouse model of breast cancer.

FIG. 7A illustrates that the DD-CT20p peptide is co-localized withmitochondria. FIG. 7B shows these observations quantified and on a bargraph.

FIGS. 8A and 8B are immunoblots that show the mitochondrialtranslocation of EGFP-tagged with Bax CT (EGFP-KK) and K189/L190 mutantsin Bax^(+/+) HCT-116 cells (FIG. 8A) and Bax^(−/−) HCT-116 cells (FIG.8B). FIGS. 8C and 8D show Bax^(+/+) HCT-116 cells (FIG. 8C) andBax^(−/−) HCT-116 cells (FIG. 8D) that were transfected with EGFP-Kk orK189/K190 mutants.

FIGS. 9A and 9B show the effect of nanoparticles alone (FIG. 9A) or withBAX peptide (FIG. 9B) on lipid vesicles.

FIG. 10 shows the expression of Bcl-2 in transiently transfectedMDA-MB-231 cells.

FIGS. 11A and B show a model for a membrane pore formed by the CT20 Baxpeptide. FIG. 11A shows peptide molecules in ribbon format. FIG. 11Bshows a top view of the pore formed by CT20p in CPK format. A calceinmolecule is shown within the pore in a ball and stick format.

FIG. 12 shows a hypothetical cell death pathway for CT20p peptide usinga basic model of apoptotic and non-apoptotic cell death.

FIG. 13 is a table of CT20p peptide comparison with antimicrobial andapoptosis inducing peptides.

FIG. 14 shows a graph of killing of microbial cells, a measure of wildtype E. coli growth by optical density (OD280) over 24 hours withincreasing concentration of CT20p. The inhibiting concentration (IC50)was found to be 50 ug of CT20p in 100 mL LB broth.

FIG. 15 shows a graph of killing microbial cells, a measure of wild typeE. coli colony formation after treatment with both CT20p and Scrambled(SCR) peptide shows that the minimum inhibitory concentration (MiC) ofthe CT20p is 25 mg. This was determined by plating the E. coli aftertreatment then counting the resulting colonies after 24 hour incubation.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention comprises methods and compositions for disruptingthe cellular membranes of cells. The cells may be prokaryotes oreukaryotes, and may be found as individual cells, in colonies, on orwithin multi-celled organisms such as plants or animals. For example,microbial cells may be found on the surface of an animal, such as ahuman, or within the animal. Cancer cells may be found in a subject,such as a plant, human or animal.

Mitochondria play a central role in regulating both apoptotic andnon-apoptotic or necrotic pathways by selectively releasing cell deathpromoting factors. The determination as to whether or not an apoptoticor non-apoptotic cell death pathway will be activated is dependent onthe type of death mediator released from the mitochondria.

Most current anti-cancer drugs only trigger the apoptotic pathway.Defects in the apoptotic machinery can contribute to tumor formation andresistance to treatment, creating a need to identify anti-cancer agentsthat kill cells by novel mechanisms. The development of a newtherapeutic agent that induces cell death via a non-apoptotic mechanismand can be used in combination with standards drugs that induceapoptosis will have significant impact on the treatment of drugresistance cancers and could greatly improve overall patient outcomes.As described herein, the death-inducing properties of the C-terminal(CT) of the alpha-9 helix of Bax, an amphipathic domain with putativemembrane binding properties, were examined.

To examine the cytotoxic potential of the CT domain of Bax, a peptide(CT20) was generated. The CT20p peptide permeabilized mitochondrial-likelipid vesicles and caused cell death. The cell death pathway triggeredby the CT20p peptide was independent of effector caspases and resistantto Bcl-2 over-expression. This indicates that the CT20p peptide can beused in combinatorial therapies to sensitize drug-resistant cancer cellsto treatment. Other properties of the CT20 Bax peptide, show that theCT20p peptide causes cell membrane permeability, and such a peptide maybe combined with antimicrobial therapeutics or therapies to cause celldeath in microorganisms, such as gram-positive and gram-negativebacteria, other types of bacteria, fungi and other microorganisms andinfectious agents.

A. Compositions

Disclosed herein are compositions comprising a CT20p peptide.

1. Compositions for Permeabilizing Membranes in Cells

Disclosed herein is a composition for permeabilizing membranes in cellscomprising a CT20p peptide. The cells may be individual cells, or cellsthat are on or in a subject. In an aspect, the cells are eukaryotic orprokaryotic cells, including but not limited to bacteria and fungi. Inan aspect, the cells are in a subject. In an aspect, the cells are on asurface, which may be inert or may be the surface of a subject. In anaspect, the cells are cancer cells or transformed cells. In an aspect,the cancer cell can be a cell from any type of cancer including, but notlimited to, cancer of the head and neck cancer, esophagus, stomach,pancreas, kidney, bladder, bone, brain, and cervix. In an aspect, thecancer is breast cancer. In an aspect, the cancer is colorectal cancer.In an aspect, the cancer is lung cancer. In an aspect, the cancer is adrug resistant cancer. In an aspect, the cancer cell is a drug resistantcancer cell. In an aspect, a disclosed composition comprising atruncated Bax peptide is administered directly into a tumor.

In an aspect, a disclosed composition for permeabilizing membranes incells forms one one or more pores in the membranes of the cells. In anaspect, the membrane may be an outer membrane, a cellular membrane or anorganelle membrane. In an aspect, the membrane is a mitochondrialmembrane. In an aspect, the one or more pores are formed in amitochondrial membrane of a cancer cell.

In an aspect, a disclosed composition for permeabilizing membranes incells destabilizes a cellular membrane. In an aspect, a disclosedcomposition facilitates ion exchange. In an aspect, a disclosedcomposition causes a sequestered molecule to be released. In an aspect,a disclosed composition destabilizes a cellular membrane, facilitatesion exchange, and causes a sequestered molecule to be released. In anaspect, a disclosed composition destabilizes a cellular membrane andfacilitates ion exchange. In an aspect, a disclosed compositiondestabilizes a cellular membrane and causes a sequestered molecule to bereleased. In an aspect, a disclosed composition facilitates ion exchangeand causes a sequestered molecule to be released.

In an aspect, a disclosed composition for permeabilizing membranes incells comprising a CT20p peptide induces cell death. In an aspect, thecell death mimics necrosis. In an aspect, the cell death occursindependent of endogenous Bax activity. In an aspect, the cell deathoccurs independent of endogenous caspase activity. In an aspect, thecell death is resistant to Bcl-2 over-expression.

In an aspect, a disclosed composition for permeabilizing membranes incells comprising a CT20p peptide induces cell death, wherein (i) thecell death mimics necrosis, (ii) the cell death occurs independent ofendogenous Bax activity, (iii) the cell death occurs independent ofendogenous caspase activity, or (iv) the cell death is resistant toBcl-2 over-expression, or (v) the cell death exhibits a combinationthereof.

In an aspect, a pore-forming composition may comprise an amount of aCT20p peptide so that pores comprising from two to ten peptides, fromtwo to eight peptides, from four to ten peptides, from five, six, seven,eight, nine, ten or more peptides, can be formed.

In an aspect, a disclosed composition for permeabilizing membranes incells comprises a CT20p peptide comprising SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, and/or SEQ ID NO: 4. For example, in an aspect, adisclosed CT20p peptide is VTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In anaspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWEEMG (SEQ ID NO:2). In an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWLLMG (SEQID NO: 3). In an aspect, a disclosed CT20p peptide isVTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosedcomposition for permeabilizing membranes in cells comprises one or moreCT20 Bax peptides, wherein the one or more CT20 Bax peptides compriseSEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4.

In an aspect, a CT20p peptide of a disclosed composition forpermeabilizing membranes in cells is encapsulated in polymericnanoparticles. In an aspect, the nanoparticles are aminated. In anaspect, the nanoparticles are carboxylated.

In an aspect, a disclosed composition for permeabilizing membranes incells comprising a CT20p peptide further comprises one or moretherapeutic compounds, such as one or more antimicrobial compounds, oneor more antibacterial compounds, one or more antifungal compounds, orone or more anti-cancer drugs, or a combination thereof. In an aspect,the one or more anti-cancer drugs comprise cisplatin. In an aspect, theone or more anti-cancer drugs induce apoptosis. In an aspect, adisclosed composition comprising a CT20p peptide further comprises oneor more chemotherapeutic drugs. In an aspect, a disclosed compositioncomprising a CT20p peptide further comprises one or moreradiosensitizers.

In an aspect, a disclosed composition for permeabilizing membranes incells comprising a CT20p peptide further comprises (i) one or moreanti-cancer drugs, (ii) one or more chemotherapeutic drugs, and (iii)one or more radiosensitizers. In an aspect, a disclosed compositionfurther comprises one or more anti-cancer drugs and one or morechemotherapeutic drugs. In an aspect, a disclosed composition furthercomprises one or more anti-cancer drugs and one or moreradiosensitizers. In an aspect, a disclosed composition furthercomprises one or more chemotherapeutic drugs and one or moreradiosensitizers.

In an aspect, a disclosed composition for permeabilizing membranes incells is administered to a subject. In an aspect, the subject is amammal. In an aspect, the mammal is a primate. In an aspect, the mammalis a human. In an aspect, the human is a patient.

In an aspect, a disclosed composition for permeabilizing membranes incells comprising a CT20p peptide is administered to a subject at leasttwo times. In an aspect, a disclosed composition is administered to thesubject two or more times. In an aspect, a disclosed composition isadministered at routine or regular intervals. For example, in an aspect,a disclosed composition is administered to the subject one time per day,or two times per day, or three or more times per day. In an aspect, adisclosed composition is administered to the subject daily, or one timeper week, or two times per week, or three or more times per week, etc.In an aspect, a disclosed composition is administered to the subjectweekly, or every other week, or every third week, or every fourth week,etc. In an aspect, a disclosed composition is administered to thesubject monthly, or every other month, or every third month, or everyfourth month, etc. In an aspect, the repeated administration of adisclosed composition occurs over a pre-determined or definite durationof time. In an aspect, the repeated administration of a disclosedcomposition occurs over an indefinite period of time.

In an aspect, following the administration of a disclosed compositionfor permeabilizing membranes in cells comprising a CT20p peptide, thecells are sensitized to treatment. In an aspect, following theadministration of a disclosed composition comprising a CT20p peptide, asubject is sensitized to treatment. In an aspect, an increasedsensitivity or a reduced sensitivity to a treatment, such as atherapeutic treatment, is measured according to one or more methods asknown in the art for the particular treatment. In an aspect, methods ofmeasuring sensitivity to a treatment include, but not limited to, cellproliferation assays and cell death assays. In an aspect, thesensitivity of a cell or a subject to treatment can be measured ordetermined by comparing the sensitivity of a cell or a subject followingadministration of a disclosed composition comprising a CT20p peptide tothe sensitivity of a cell or subject that has not been administered adisclosed composition comprising a CT20p peptide.

For example, in an aspect, following the administration of a disclosedcomposition for permeabilizing membranes in cells comprising a CT20ppeptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, more sensitiveto treatment than a cell that has not been administered a disclosedcomposition comprising a CT20p peptide. In an aspect, following theadministration of a disclosed composition comprising a CT20p peptide thecell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold,18-fold, 19-fold, 20-fold, or greater, less resistant to treatment thana cell that has not been administered a disclosed composition comprisinga CT20p peptide. The determination of a cell's or a subject'ssensitivity or resistance is routine in the art and within the skill ofan ordinary clinician and/or researcher.

In an aspect, the determination of a cell's or a subject's sensitivityor resistance to treatment can be monitored. For example, in an aspect,data regarding sensitivity or resistance resistance can be acquiredperiodically, such as every week, every other week, every month, everyother month, every 3 months, 6 months, 9 months, or every year, everyother year, every 5 years, every 10 years for the life of the subject,for example, a human subject or patient with cancer and/or aberrant cellgrowth. In an aspect, data regarding sensitivity or resistance can beacquired at various rather than at periodic times. In an aspect,treatment for a a subject can be modified based on data regarding acell's or a subject's sensitivity or resistance to treatment. Forexample, in an aspect, the treatment can modified by changing the thedose of a disclosed compositions, the route of administration of adisclosed compositions, the frequency of administration of a disclosedcomposition, etc.

Disclosed herein is a composition for permeabilizing lipid membranes incells comprising a CT20p peptide and one or more anti-cancer drugs.

2. Compositions for Killing Cells

Disclosed herein is a composition for killing cells comprising a CT20ppeptide. In an aspect, the cells are eukaryotic or prokaryotic cells,including but not limited to bacteria and fungi. In an aspect, the cellsare in a subject. In an aspect, the cells are on a surface, which may beinert or may be the surface of a subject. In an aspect, the cells arecancer cells or transformed cells. In an aspect, the cancer cell can bea cell from any type of cancer including, but not limited to, cancer ofthe head and neck cancer, esophagus, stomach, pancreas, kidney, bladder,bone, brain, and cervix. In an aspect, the cancer is breast cancer. Inan aspect, the cancer is colorectal cancer. In an aspect, the cancer islung cancer. In an aspect, the cancer is a drug resistant cancer. In anaspect, the cancer cell is a drug resistant cancer cell. In an aspect, adisclosed composition comprising a CT20p peptide is administereddirectly into a tumor.

In an aspect, a disclosed composition for killing cells forms one ormore pores in the membranes of the cells. In an aspect, the membrane maybe an outer membrane, a cellular membrane or an organelle membrane. Inan aspect, the membrane is a mitochondrial membrane. In an aspect, theone or more pores are formed in a mitochondrial membrane. In an aspect,the one or more pores are formed in a mitochondrial membrane of a cancercell.

In an aspect, a disclosed composition for killing cells destabilizes acellular membrane. In an aspect, a disclosed composition facilitates ionexchange. In an aspect, a disclosed composition causes a sequesteredmolecule to be released. In an aspect, a disclosed compositiondestabilizes a cellular membrane, facilitates ion exchange, and causes asequestered molecule to be released. In an aspect, a disclosedcomposition destabilizes a cellular membrane and facilitates ionexchange. In an aspect, a disclosed composition destabilizes a cellularmembrane and causes a sequestered molecule to be released. In an aspect,a disclosed composition facilitates ion exchange and causes asequestered molecule to be released.

In an aspect, a disclosed composition for killing cells comprising aCT20p peptide induces cell death. In an aspect, the cell death mimicsnecrosis. In an aspect, the cell death occurs independent of endogenousBax activity. In an aspect, the cell death occurs independent ofendogenous caspase activity. In an aspect, the cell death is resistantto Bcl-2 over-expression.

In an aspect, a disclosed composition for killing cells comprising aCT20p peptide induces cell death, wherein (i) the cell death mimicsnecrosis, (ii) the cell death occurs independent of endogenous Baxactivity, (iii) the cell death occurs independent of endogenous caspaseactivity, or (iv) the cell death is resistant to Bcl-2 over-expression,or (v) the cell death exhibits a combination thereof.

In an aspect, a composition for killing cells comprises a pore-formingcomposition which may comprise an amount of a CT20p peptide so thatpores comprising from two to ten peptides, from two to eight peptides,from four to ten peptides, from five, six, seven, eight, nine, ten ormore peptides, can be formed.

In an aspect, a disclosed composition for killing cells comprises aCT20p peptide comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,and/or SEQ ID NO: 4. For example, in an aspect, a disclosed CT20ppeptide peptide is VTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, adisclosed CT20p peptideis VTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In anaspect, a disclosed CT20p peptideis VTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3).In an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWRRMG (SEQ IDNO: 4). In an aspect, a disclosed composition for killing cellscomprises one or more CT20 Bax peptides, wherein the one or more CT20Bax peptides comprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ IDNO: 4.

In an aspect, a CT20p peptide of a disclosed composition for killingcells is encapsulated in polymeric nanoparticles. In an aspect, thenanoparticles are aminated. In an aspect, the nanoparticles arecarboxylated.

In an aspect, a disclosed composition for killing cells comprising aCT20p peptide further comprises one or more therapeutic compounds, suchas one or more antimicrobial compounds, one or more antibacterialcompounds, one or more antifungal compounds, or one or more anti-cancerdrugs, or a combination thereof. In an aspect, the one or moreanti-cancer drugs comprise cisplatin. In an aspect, the one or moreanti-cancer drugs induce apoptosis. In an aspect, a disclosedcomposition for killing cells comprising a CT20p peptide furthercomprises one or more chemotherapeutic drugs. In an aspect, a disclosedcomposition for killing cells comprising a CT20p peptide furthercomprises one or more radiosensitizers.

In an aspect, a disclosed composition for killing cells comprising aCT20p peptide further comprises (i) one or more anti-cancer drugs, (ii)one or more chemotherapeutic drugs, and (iii) one or moreradiosensitizers. In an aspect, a disclosed composition for killingcells further comprises one or more anti-cancer drugs and one or morechemotherapeutic drugs. In an aspect, a disclosed composition forkilling cells further comprises one or more anti-cancer drugs and one ormore radiosensitizers. In an aspect, a disclosed composition for killingcells further comprises one or more chemotherapeutic drugs and one ormore radiosensitizers.

In an aspect, a disclosed composition for killing cells is administeredto a subject. In an aspect, the subject is a mammal. In an aspect, themammal is a primate. In an aspect, the mammal is a human. In an aspect,the human is a patient.

In an aspect, a disclosed composition for killing cells comprising aCT20p peptide is administered to a subject at least two times. In anaspect, a disclosed composition is administered to the subject two ormore times. In an aspect, a disclosed composition is administered atroutine or regular intervals. For example, in an aspect, a disclosedcomposition is administered to the subject one time per day, or twotimes per day, or three or more times per day. In an aspect, a disclosedcomposition is administered to the subject daily, or one time per week,or two times per week, or three or more times per week, etc. In anaspect, a disclosed composition is administered to the subject weekly,or every other week, or every third week, or every fourth week, etc. Inan aspect, a disclosed composition is administered to the subjectmonthly, or every other month, or every third month, or every fourthmonth, etc. In an aspect, the repeated administration of a disclosedcomposition occurs over a pre-determined or definite duration of time.In an aspect, the repeated administration of a disclosed compositionoccurs over an indefinite period of time.

In an aspect, following the administration of a disclosed compositionfor killing cells comprising a CT20p peptide, the cells are sensitizedto treatment. In an aspect, following the administration of a disclosedcomposition for killing cells comprising a CT20p peptide, a subject issensitized to treatment. In an aspect, an increased sensitivity or areduced sensitivity sensitivity to a treatment, such as a therapeutictreatment, is measured according to one or more methods as known in theart for the particular treatment. In an aspect, methods of measuringsensitivity to a treatment include, but not limited to, cellproliferation assays and cell death assays. In an aspect, thesensitivity of a cell or a subject to treatment can be measured ordetermined by comparing the sensitivity of a cell or a subject followingadministration of a disclosed composition for killing cells comprising aCT20p peptide to the sensitivity of a cell or subject that has not beenadministered a disclosed composition for killing cells comprising aCT20p peptide.

For example, in an aspect, following the administration of a disclosedcomposition for killing cells comprising a CT20p peptide, the cell is2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,19-fold, 20-fold, or greater, more sensitive to treatment than a cellthat has not been administered a disclosed composition for killing cellscomprising a CT20p peptide. In an aspect, following the administrationof a disclosed composition for killing cells comprising a CT20p peptide,the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold,17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant totreatment than a cell that has not been administered a disclosedcomposition for killing cells comprising a CT20p peptide. Thedetermination of a cell's or a subject's sensitivity or resistance isroutine in the art and within the skill of an ordinary clinician and/orresearcher.

In an aspect, the determination of a cell's or a subject's sensitivityor resistance to treatment can be monitored. For example, in an aspect,data regarding sensitivity or resistance can be acquired periodically,such as every week, every other week, every month, every other month,every 3 months, 6 months, 9 months, or every year, every other year,every 5 years, every 10 years for the life of the subject, for example,a human subject or patient with cancer and/or aberrant cell growth. Inan aspect, data regarding sensitivity or resistance can be acquired atvarious rather than at periodic times. In an aspect, treatment for asubject can be modified based on data regarding a cell's or a subject'ssensitivity or resistance to treatment. For example, in an aspect, thetreatment can modified by changing the dose of a disclosed compositions,the route of administration of a disclosed compositions, the frequencyof administration of a disclosed composition, etc.

Disclosed herein is a composition for killing cells comprising a CT20ppeptide and one or more anti-cancer drugs.

3. Pharmaceutical Compositions

In an aspect, the invention relates to pharmaceutical compositionscomprising a disclosed composition for permeabilizing membranes incells. In an aspect, the invention relates to pharmaceuticalcompositions comprising a disclosed composition for killing cells. In anaspect, the disclosed compositions for permeabilizing membranes andcells and for killing cells comprise a CT20 Bax peptide. In an aspect, apharmaceutical composition can be provided comprising a therapeuticallyeffective amount of at least one disclosed composition and apharmaceutically acceptable carrier.

B. Methods Comprising a Disclosed Composition 1. PermeabilizingMembranes of Cells

Disclosed herein are methods of permeabilizing membranes of cells. Thecells may be individual cells, or cells that are on or in a subject. Inan aspect, the cells are eukaryotic or prokaryotic cells, including butnot limited to bacteria and fungi.

In an aspect, disclosed herein is a method of permeabilizing membranesof cells in a subject comprising administering to at least a cell aCT20p peptide. In an aspect, disclosed herein is a method ofpermeabilizing membranes of cells in a subject comprising administeringto at least a cell a composition comprising an effective amount of aCT20p peptide. In an aspect, the membrane may be an outer membrane, acellular membrane or an organelle membrane. In an aspect the cell is abacterial cell. In an aspect, the cell is a fungal cell. In an aspect,the cell is a microbial cell. In an aspect, the cell is a cancer cell ora transformed cell. In an aspect, the cancer cell can be a cell from anytype of cancer including, but not limited to, cancer of the head andneck cancer, esophagus, stomach, pancreas, kidney, bladder, bone, brain,and cervix. In an aspect, the cancer is breast cancer. In an aspect, thecancer is colorectal cancer. In an aspect, the cancer is lung cancer. Inan aspect, the cancer is a drug resistant cancer. In an aspect, thecancer cell is a drug resistant cancer cell. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide is administered directly into a tumor.

In an aspect of a disclosed a method of permeabilizing membranes ofcells, a CT20p peptide or a composition comprising an effective amountof a CT20p peptide forms at least one pore in the membrane of the cell.In an aspect, a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide forms two or more pores in the membrane of thecell. In an aspect, cell is a cancer cell. In an aspect, the one or morepores are formed in a mitochondrial membrane. In an aspect, the one ormore pores are formed in a mitochondrial membrane of a cancer cell.

In an aspect of a disclosed method of permeabilizing membranes of cellsin a subject, a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide destabilizes a cellular membrane, facilitatesion exchange, and causes a sequestered molecule to be released. In anaspect, a CT20p peptide or a composition comprising an effective amountof a CT20p peptide destabilizes a cellular membrane. In an aspect, aCT20p peptide or a composition comprising an effective amount of a CT20ppeptide facilitates ion exchange, exchange. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide causes a sequestered molecule to be released. In an aspect, aCT20p peptide or a composition comprising an effective amount of a CT20ppeptide destabilizes a cellular membrane and facilitates ion exchange.In an aspect, a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide destabilizes a cellular membrane and causes asequestered molecule to be released. In an aspect, a CT20p peptide or acomposition comprising an effective amount of a CT20p peptidefacilitates ion exchange and causes a sequestered molecule to bereleased.

In an aspect, following the administration of a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide, the cellsare sensitized to treatment. In an aspect, following the administrationof CT20p peptide or a composition comprising an effective amount of aCT20p peptide, a subject is sensitized to treatment. In an aspect, anincreased sensitivity or a reduced sensitivity to a treatment, such as atherapeutic treatment, is measured according to one or more methods asknown in the art for the particular treatment. In an aspect, methods ofmeasuring sensitivity to a treatment include, but not limited to, cellproliferation assays and cell death assays. In an aspect, thesensitivity of a cell or a subject to treatment can be measured ordetermined by comparing the sensitivity of a cell or a subject followingadministration of a CT20p peptide or a composition comprising aneffective amount of a CT20p peptide to the sensitivity of a cell orsubject that has not been administered a CT20p peptide or a compositioncomprising an effective amount of a CT20p peptide.

For example, in an aspect, following the administration of a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, more sensitiveto treatment than a cell that has not been administered a CT20p peptideor a disclosed composition comprising an effective amount of a CT20ppeptide. In an aspect, following the administration of a CT20p peptideor a composition comprising an effective amount of a CT20p peptide, thecell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold,18-fold, 19-fold, 20-fold, or greater, less resistant to treatment thana cell that has not been administered a CT20p peptide or a disclosedcomposition comprising an effective amount of a CT20p peptide. Thedetermination of a cell's or a subject's sensitivity or resistance isroutine in the art and within the skill of an ordinary clinician and/orresearcher.

In an aspect, the determination of a cell's or a subject's sensitivityor resistance to treatment can be monitored. For example, in an aspect,data regarding sensitivity or resistance can be acquired periodically,such as every week, every other week, every month, every other month,every 3 months, 6 months, 9 months, or every year, every other year,every 5 years, every 10 years for the life of the subject, for example,a human subject or patient with cancer and/or aberrant cell growth. Inan aspect, data regarding sensitivity or resistance can be acquired atvarious rather than at periodic times. In an aspect, treatment for asubject can be modified based on data regarding a cell's or a subject'ssensitivity or resistance to treatment. For example, in an aspect, thetreatment can modified by changing the dose of a disclosed compositions,the route of administration of a disclosed compositions, the frequencyof administration of a disclosed composition, etc.

In an aspect, a disclosed method of permeabilizing membranes of cells ina subject further comprises repeating the administration a CT20p peptideor a composition comprising an effective amount of a CT20p peptide. Inan aspect, a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide is administered to the subject at least twotimes. In an aspect, a CT20p peptide or a composition comprising aneffective amount of a CT20p peptide is administered to the subject twoor more times. In an aspect, a CT20p peptide or a composition comprisingan effective amount of a CT20p peptide administered at routine orregular intervals. For example, in an aspect, a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide isadministered to the subject one time per day, or two times per day, orthree or more times per day. In an aspect, a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide isadministered to the subject daily, or one time per week, or two timesper week, or three or more times per week, etc. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide is administered to the subject weekly, or every other week, orevery third week, or every fourth week, etc. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide is administered to the subject monthly, or every other month, orevery third month, or every fourth month, etc. In an aspect, therepeated administration of a CT20p peptide or a composition comprisingan effective amount of a CT20p peptide occurs over a pre-determined ordefinite duration of time. In an aspect, the repeated administration ofa CT20p peptide or a composition comprising an effective amount of aCT20p peptide occurs over an indefinite period of time.

In an aspect, a disclosed method of permeabilizing membranes of cells ina subject further comprises inducing cell death. In an aspect, celldeath mimics necrosis. In an aspect, cell death occurs independent ofendogenous Bax activity. In an aspect, cell death occurs independent ofendogenous caspase activity. In an aspect, cell death is resistant toBcl-2 over-expression.

In an aspect, a disclosed method of permeabilizing membranes of cells ina subject comprising administering a CT20p peptide or a compositioncomprising an effective amount of a CT20p peptide further comprisesinducing cell death. Cell death may include, but is not limited towherein (i) cell death mimics necrosis, (ii) cell death occursindependent of endogenous Bax activity, (iii) cell death occursindependent of endogenous caspase activity, or (iv) cell death isresistant to Bcl-2 over-expression, or (v) cell death exhibits acombination thereof.

In an aspect, a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide of a disclosed method of permeabilizing cellmembranes comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQID NO: 4. For example, in an aspect, a CT20p peptide isVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosedcomposition comprising an effective amount of a CT20p peptide comprisesone or more CT20 Bax peptides, wherein the one or more CT20 Bax peptidescomprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4, or twoor more of each. In an aspect, a CT20p peptide of a disclosed method ofpermeabilizing membranes of cells in a subject is encapsulated inpolymeric nanoparticles. In an aspect, the nanoparticles are aminated.In an aspect, the nanoparticles are carboxylated.

In an aspect, a disclosed method of permeabilizing membranes comprisingadministering a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide further comprises administering one or moreanti-cancer drugs. In an aspect, the one or more anti-cancer drugscomprise cisplatin. In an aspect, the one or more anti-cancer drugsinduce apoptosis. In an aspect, a disclosed method of permeabilizingmembranes comprising administering a CT20p peptide or a compositioncomprising an effective amount of a CT20p peptide further comprisesadministering one or more chemotherapeutic drugs. In an aspect, adisclosed method of permeabilizing membranes comprising administering aCT20p peptide or or a composition comprising an effective amount of aCT20p peptide further comprises administering one or moreradiosensitizers.

In an aspect, a disclosed method of permeabilizing membranes comprisingadministering a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide further comprises administering (i) one ormore anti-cancer drugs, (ii) one or more chemotherapeutic drugs, and(iii) one or more radiosensitizers. In an aspect, a disclosed methodfurther comprises administering one or more anti-cancer drugs and one ormore chemotherapeutic drugs. In an aspect, a disclosed method furthercomprises administering one or more anti-cancer drugs and one or moreradiosensitizers. In an aspect, a disclosed method further comprisesadministering one or more chemotherapeutic drugs and one or moreradiosensitizers.

In an aspect of a disclosed method of permeabilizing membranes of cells,which may be in or on a subject, the subject is a mammal. In an aspect,the mammal is a primate. In an aspect, the mammal is a human. In anaspect, the human is a patient.

In an aspect, disclosed herein is a method of permeabilizing membranesof cells in a subject comprising (i) administering to a subject aneffective amount of a CT20p peptide or a composition comprising aneffective amount of a CT20p peptide, and (ii) forming at least one porein a membrane of at least one cell of the subject, wherein the peptidedestabilizes at least one membrane, facilitates ion exchange, and/orcauses a sequestered molecule to be released.

2. Killing Cancer Cells

Disclosed herein are methods of killing cancer cells.

In an aspect, disclosed herein is a method of killing cancer cells in asubject comprising administering to at least one cell of a subject aneffective amount of a CT20p peptide. In an aspect, disclosed herein is amethod of killing cancer cells in a subject comprising administering toat least one cell of a subject a composition comprising an effectiveamount of a CT20p peptide. In an aspect, the cell is a cancer cell or atransformed cell. In an aspect, the cancer cell can be a cell from anytype of cancer including, but not limited to, cancer of the head andneck cancer, esophagus, stomach, pancreas, kidney, bladder, bone, brain,and cervix. In an aspect, the cancer is breast cancer. In an aspect, thecancer is colorectal cancer. In an aspect, the cancer is lung cancer. Inan aspect, the cancer is a drug resistant cancer. In an aspect, thecancer cell is a drug resistant cancer cell. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide is administered directly into a tumor.

In an aspect, a disclosed method of killing cancer cells in a subjectfurther comprises permeabilizing at least one membrane in the cell. Inan aspect, the membrane is a mitochondrial membrane. In an aspect, theone or more pores are formed in a mitochondrial membrane. In an aspect,the one or more pores are formed in a mitochondrial membrane of a cancercell.

In an aspect, permeabilizing at least one membrane in the cell comprisesdestabilizing a cellular membrane, facilitating ion exchange, andcausing a sequestered molecule to be released. In an aspect,permeabilizing at least one membrane in the cell comprises destabilizesa cellular membrane. In an aspect, permeabilizing at least one membranein the cell comprises facilitating ion exchange. In an aspect,permeabilizing at least one membrane in the cell comprises causing asequestered molecule to be released. In an aspect, permeabilizing atleast one membrane in the cell comprises destabilizing a cellularmembrane and facilitating ion exchange. In an aspect, permeabilizing atleast one membrane in the cell comprises destabilizing a cellularmembrane and causing a sequestered molecule to be released. In anaspect, permeabilizing at least one membrane in the cell comprisesfacilitating ion exchange and causing a sequestered molecule to bereleased.

In an aspect of a disclosed method of killing cancer cells in a subject,the cancer cells are sensitized to treatment. In an aspect of adisclosed method of killing cancer cells in a subject, the subject issensitized to treatment. In an aspect, an increased sensitivity or areduced sensitivity to a treatment, such as a therapeutic treatment, ismeasured according to one or more methods as known in the art for theparticular treatment. In an aspect, methods of measuring sensitivity toa treatment include, but not limited to, cell proliferation assays andcell death assays. In an aspect, the sensitivity of a cell or a subjectto treatment can be measured or determined by comparing the sensitivityof a cell or a subject following administration of a CT20p peptide or acomposition comprising a CT20p peptide to the sensitivity of a cell orsubject that has not been administered a CT20p peptide or a compositioncomprising a CT20p peptide.

For example, in an aspect, following the administration of a CT20ppeptide or a composition comprising a CT20p peptide, the cell is 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,19-fold, 20-fold, or greater, more sensitive to treatment than a cellthat has not been administered a CT20p peptide or a compositioncomprising a CT20p peptide. In an aspect, following the administrationof a CT20p peptide or a composition comprising a CT20p peptide, the cellis 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold,18-fold, 19-fold, 20-fold, or greater, less resistant to treatment thana cell that has not been administered a CT20p peptide or a compositioncomprising a CT20p peptide. The determination of a cell's or a subject'ssensitivity or resistance is routine in the art and within the skill ofan ordinary clinician and/or researcher.

In an aspect, the determination of a cell's or a subject's sensitivityor resistance to treatment can be monitored. For example, in an aspect,data regarding sensitivity or resistance can be acquired periodically,such as every week, every other week, every month, every other month,every 3 months, 6 months, 9 months, or every year, every other year,every 5 years, every 10 years for the life of the subject, for example,a human subject or patient with cancer and/or aberrant cell growth. Inan aspect, data regarding sensitivity or resistance can be acquired atvarious rather than periodic times. In an aspect, treatment for asubject can be modified based on data regarding a cell's or a subject'ssensitivity or resistance to treatment.

In an aspect, a disclosed method of killing cancer cells in a subjectfurther comprises repeating the administration of a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide. In anaspect, a CT20p peptide or a composition comprising an effective amountof a CT20p peptide is administered to the subject at least two times. Inan aspect, a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide is administered to the subject two or moretimes. In an aspect, a CT20p peptide or a composition comprising aneffective amount of a CT20p peptide is administered at routine orregular intervals. For example, in an aspect, a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide isadministered to the subject one time per day, or two times per day, orthree or more times per day. In an aspect, a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide isadministered to the subject daily, or one time per week, or two timesper week, or three or more times per week, etc. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide is administered to the subject weekly, or every other week, orevery third week, or every fourth week, etc. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide is administered to the subject monthly, or every other month, orevery third month, or every fourth month, etc. In an aspect, therepeated administration of a CT20p peptide or a composition comprisingan effective amount of a CT20p peptide occurs over a pre-determined ordefinite duration of time. In an aspect, the repeated administration ofa CT20p peptide or a composition comprising an effective amount of aCT20p peptide occurs over an indefinite period of time.

In an aspect, a disclosed method of killing cancer cells in a subjectfurther comprises inducing cell death. In an aspect, cell death mimicsnecrosis. In an aspect, cell death occurs independent of endogenous Baxactivity. In an aspect, cell death occurs independent of endogenouscaspase activity. In an aspect, cell death is resistant to Bcl-2over-expression. In an aspect, a disclosed method of killing cancercells induces cell death, wherein (i) cell death mimics necrosis, (ii)cell death occurs independent of endogenous Bax activity, (iii) celldeath occurs independent of endogenous caspase activity, or (iv) celldeath is resistant to Bcl-2 over-expression, or (v) cell death exhibitsa combination thereof.

In an aspect, a CT20p peptide of a disclosed method of killing cellscomprises SEQ ID ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO:4. For example, in an aspect, a C-terminal truncated Bax isVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosedcomposition comprising an effective amount of a CT20p peptide comprisesone or more CT20 Bax peptides, wherein the one or more CT20 Bax peptidescomprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4.

In an aspect, a CT20p peptide of a disclosed method of killing cancercells in a subject is encapsulated in polymeric nanoparticles. In anaspect, the nanoparticles are aminated. In an aspect, the nanoparticlesare carboxylated.

In an aspect, a disclosed method for killing cancer cells comprisingadministering a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide further comprises administering one or moreanti-cancer drugs. In an aspect, the one or more anticancer drugscomprise cisplatin. In an aspect, the one or more anti-cancer drugsinduce apoptosis. In an aspect, a disclosed method for killing cancercells comprising administering a CT20p peptide or a compositioncomprising an effective amount of a CT20p peptide further comprisesadministering one or more chemotherapeutic drugs. In an aspect, adisclosed method for killing cancer cells comprising administering aCT20p peptide or a composition comprising an effective amount of a CT20ppeptide further comprises administering one or more radiosensitizers.

In an aspect, a disclosed method for killing cancer cells comprisingadministering a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide further comprises administering (i) one ormore anti-cancer drugs, (ii) one or more chemotherapeutic drugs, and(iii) one or more radiosensitizers. In an aspect, a disclosed method forkilling cancer cells further comprises administering one or moreanti-cancer drugs and one or more chemotherapeutic drugs. In an aspect,a disclosed method for killing cancer cells further comprisesadministering one or more anti-cancer drugs and one or moreradiosensitizers. In an aspect, a disclosed method for killing cancercells further comprises administering one or more chemotherapeutic drugsand one or more radiosensitizers.

In an aspect of a disclosed method of killing cancer cells in a subject,the subject is a mammal. In an aspect, the mammal is a primate. In anaspect, the mammal is a human. In an aspect, the human is a patient.

Disclosed herein is a method of killing cancer cells in a subjectcomprising (i) administering to a subject an effective amount of a CT20ppeptide, (ii) permeabilizing at least one membrane in a cell of thesubject, and (iii) inducing cell death.

3. Killing Microbial Cells

Disclosed herein are methods of killing microbial cells.

In an aspect, disclosed herein is a method of killing microbial cells ina subject comprising administering to at least one cell of a subject aneffective amount of a CT20p peptide. In an aspect, disclosed herein is amethod of killing microbial cells in a subject comprising administeringto at least one cell of a subject a composition comprising an effectiveamount of a CT20p peptide. In an aspect, the cell is a microbial cell,including eukaryotic and prokaryotic cells, such as bacteria and/orfungi. In an aspect, the cell is a drug resistant microbial cell. In anaspect, a CT20p peptide or a composition comprising an effective amountof a CT20p peptide is administered directly.

In an aspect, a disclosed method of killing microbial cells in a subjectfurther comprises permeabilizing at least one membrane in the cell. Inan aspect, the membrane is a mitochondrial membrane. In an aspect, theone or more pores are formed in a mitochondrial membrane. In an aspect,the one or more pores are formed in a mitochondrial membrane of amicrobial cell.

In an aspect, permeabilizing at least one membrane in the cell comprisesdestabilizing a cellular membrane, facilitating ion exchange, andcausing a sequestered molecule to be released. In an aspect,permeabilizing at least one membrane in the cell comprises destabilizesa cellular membrane. In an aspect, permeabilizing at least one membranein the cell comprises facilitating ion exchange. In an aspect,permeabilizing at least one membrane in the cell comprises causing asequestered molecule to be released. In an aspect, permeabilizing atleast one membrane in the cell comprises destabilizing a cellularmembrane and facilitating ion exchange. In an aspect, permeabilizing atleast one membrane in the cell comprises destabilizing a cellularmembrane and causing a sequestered molecule to be released. In anaspect, permeabilizing at least one membrane in the cell comprisesfacilitating ion exchange and causing a sequestered molecule to bereleased.

In an aspect of a disclosed method of killing microbial cells in asubject, the microbial cells are sensitized to treatment. In an aspectof a disclosed method of killing microbial cells in a subject, thesubject is sensitized to treatment. In an aspect, an increasedsensitivity or a reduced sensitivity to a treatment, such as atherapeutic treatment, is measured according to one or more methods asknown in the art for the particular treatment. In an aspect, methods ofmeasuring sensitivity to a treatment include, but not limited to, cellproliferation assays and cell death assays. In an aspect, thesensitivity of a cell or a subject to treatment can be measured ordetermined by comparing the sensitivity of a cell or a subject followingadministration of a CT20p peptide or a composition comprising a CT20ppeptide to the sensitivity of a cell or subject that has not beenadministered a CT20p peptide or a composition comprising a CT20ppeptide.

For example, in an aspect, following the administration of a CT20ppeptide or a composition comprising a CT20p peptide, the cell is 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,19-fold, 20-fold, or greater, more sensitive to treatment than a cellthat has not been administered a CT20p peptide or a compositioncomprising a CT20p peptide. In an aspect, following the administrationof a CT20p peptide or a composition comprising a CT20p peptide, the cellis 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold,18-fold, 19-fold, 20-fold, or greater, less resistant to treatment thana cell that has not been administered a CT20p peptide or a compositioncomprising a CT20p peptide. The determination of a cell's or a subject'ssensitivity or resistance is routine in the art and within the skill ofan ordinary clinician and/or researcher.

In an aspect, the determination of a cell's or a subject's sensitivityor resistance to treatment can be monitored. For example, in an aspect,data regarding sensitivity or resistance can be acquired periodically,such as every week, every other week, every month, every other month,every 3 months, 6 months, 9 months, or every year, every other year,every 5 years, every 10 years for the life of the subject, for example,a human subject. In an aspect, data regarding sensitivity or resistancecan be acquired at various rather than periodic times. In an aspect,treatment for a subject can be modified based on data regarding a cell'sor a subject's sensitivity or resistance to treatment.

In an aspect, a disclosed method of killing microbial cells in a subjectfurther comprises repeating the administration of a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide. In anaspect, a CT20p peptide or a composition comprising an effective amountof a CT20p peptide is administered to the subject at least two times. Inan aspect, a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide is administered to the subject two or moretimes. In an aspect, a CT20p peptide or a composition comprising aneffective amount of a CT20p peptide is administered at routine orregular intervals. For example, in an aspect, a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide isadministered to the subject one time per day, or two times per day, orthree or more times per day. In an aspect, a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide isadministered to the subject daily, or one time per week, or two timesper week, or three or more times per week, etc. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide is administered to the subject weekly, or every other week, orevery third week, or every fourth week, etc. In an aspect, a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide is administered to the subject monthly, or every other month, orevery third month, or every fourth month, etc. In an aspect, therepeated administration of a CT20p peptide or a composition comprisingan effective amount of a CT20p peptide occurs over a pre-determined ordefinite duration of time. In an aspect, the repeated administration ofa CT20p peptide or a composition comprising an effective amount of aCT20p peptide occurs over an indefinite period of time.

In an aspect, a disclosed method of killing microbial cells in a subjectfurther comprises inducing cell death. In an aspect, cell death mimicsnecrosis. In an aspect, cell death occurs independent of endogenous Baxactivity. In an aspect, cell death occurs independently of endogenouscaspase activity. In an aspect, cell death is resistant to Bcl-2over-expression. In an aspect, a disclosed method of killing cellsinduces cell death, wherein (i) cell death mimics necrosis, (ii) celldeath occurs independent of endogenous Bax activity, (iii) cell deathoccurs independent of endogenous caspase activity, or (iv) cell death isresistant to Bcl-2 over-expression, or (v) cell death exhibits acombination thereof.

In an aspect, a CT20p peptide of a disclosed method of killing cellscomprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4.For example, in an aspect, a C-terminal truncated Bax isVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3). In an aspect, a CT20p peptide isVTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosedcomposition comprising an effective amount of a CT20p peptide comprisesone or more CT20 Bax peptides, wherein the one or more CT20 Bax peptidescomprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4.

In an aspect, a CT20p peptide of a disclosed method of killing microbialcells in a subject is encapsulated in polymeric nanoparticles. In anaspect, the nanoparticles are aminated. In an aspect, the nanoparticlesare carboxylated.

In an aspect, a disclosed method for killing microbial cells comprisingadministering a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide further comprises administering one or moreanti-microbial drugs. In an aspect, a disclosed method for killingmicrobial cells comprising administering a CT20p peptide or acomposition comprising an effective amount of a CT20p peptide furthercomprises administering one or more chemotherapeutic drugs. In anaspect, a disclosed method for killing cancer cells comprisingadministering a CT20p peptide or a composition comprising an effectiveamount of a CT20p peptide further comprises administering one or moreantimicrobial therapeutics or therapies.

In an aspect of a disclosed method of killing microbial cells in or on asubject, the subject is a mammal. In an aspect, the mammal is a primate.In an aspect, the mammal is a human. In an aspect, the human is apatient.

Disclosed herein is a method of killing microbial cells in a subjectcomprising (i) administering to a subject an effective amount of a CT20ppeptide, (ii) permeabilizing at least one membrane in a cell of thesubject, and (iii) inducing cell death.

4. Other Uses

Also disclosed herein are uses of a disclosed composition as aninvestigational and/or research tool in the development andstandardization of in vitro and in vivo test systems for evaluation inlaboratory animals such as cats, dogs, rabbits, monkeys, rats and mice,as part of the search for (i) new therapeutic approaches forpermeabilizing membranes of cells and for killing cancer or microbialcells as well as (ii) the evaluation of the permeabilization ofmembranes of cells and for the killing of cancer or microbial cells. Inan aspect, the search for new therapeutic approaches and the evaluationof new therapeutic approaches involves a subject, such as a humansubject or human patient.

Methods of killing microbial organisms on an inert surface.

The present invention comprises methods of treating a surface to renderit antimicrobial, comprising contacting a CT20p peptide compositioncomprising CT20p peptide to a surface. The peptides may be air-dried andremain on the surface or may be affixed to the surface by binding toother proteins or binding-partners on the surface, or may be attached byadhesive compounds or other compounds known for attaching proteins to asurface, or the peptides may remain in solution in a liquid or semisolidor other form that contacts the surface. The CT20p peptide compositionmay comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO:4, or a combination of two or more of SEQ ID NOs 1-4. When a surfacetreated with a CT20p peptide composition comprising one or more CT20ppeptides is then contacted by microbial organisms, it is believed thatthe cellular membranes of the microbial organisms are permeablized andmicrobial death may occur.

C. Definitions

Unless otherwise expressly stated, it is in no way intended that anymethod or aspect set forth herein be construed as requiring that itssteps be performed in a specific order. Accordingly, where a methodclaim does not specifically state in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including matters of logic withrespect to arrangement of steps or operational flow, plain meaningderived from grammatical organization or punctuation, or the number ortype of aspects described in the specification.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

Ranges can be expressed herein as from “about” one particular value,and/or to to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is is also understood that each unit between twoparticular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, the amino acid abbreviations are conventional one lettercodes for the amino acids and are expressed as follows: A, alanine; B,asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate,glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine;K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q,glutamine; R, arginine; S, serine; T, threonine; V, valine; W,tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

“Peptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. For example,a peptide can be a fragment of a full-length protein, such as, forexample, the CT20 Bax peptide. A peptide is comprised of consecutiveamino acids. The term “peptide” encompasses naturally occurring orsynthetic molecules.

In general, the biological activity or biological action of a peptiderefers to any function exhibited or performed by the peptide that isascribed to the naturally occurring form of the peptide as measured orobserved in vivo (i.e., in the natural physiological environment of theprotein) or in vitro (i.e., under laboratory conditions). For example, abiological activity of the CT20p peptide is the cytotoxic activity ofthe CT20 Bax peptide.

The term “enzyme” as used herein refers to any peptide that catalyzes achemical reaction of other substances without itself being destroyed oraltered upon completion of the reaction. Typically, a peptide havingenzymatic activity catalyzes the formation of one or more products fromone or more substrates. Such peptides can have any type of enzymaticactivity including, without limitation, the enzymatic activity orenzymatic activities associated with enzymes such as those disclosedherein.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or can not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the terms “transformation” and “transfection” mean theintroduction of a nucleic acid, e.g., an expression vector, into arecipient cell including introduction of a nucleic acid to thechromosomal DNA of said cell. The art is familiar with variouscompositions, methods, techniques, etc. used to effect the introductionof a nucleic acid into a recipient cell. The art is familiar with suchcompositions, methods, techniques, etc for both eukaryotic andprokaryotic cells. The art is familiar with such compositions, methods,techniques, etc. for the optimization of the introduction and expressionof a nucleic acid into and within a recipient cell.

As used herein, “a CT20p peptide” may refer to one peptide or may referone or more peptides, such as molar concentrations of the peptide, aswould be found in a composition. Those skilled in the art understandwhere an individual peptide is intended and where a molar, or smaller orlarger amount, of many of the same peptide are intended.

As used herein, the term “subject” refers to the target ofadministration, e.g., an animal. Thus, the subject of the hereindisclosed methods can be a vertebrate, such as a mammal, a fish, a bird,a reptile, or an amphibian. Alternatively, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a patient. A patient refers to a subjectafflicted with a disease or disorder, such as, for example, cancerand/or aberrant cell growth. The term “patient” includes human andveterinary subjects. In some aspects of the disclosed methods, thesubject has been diagnosed with a need for treatment for cancer and/oraberrant cell growth.

Therapeutic agents may include antimicrobial agents, such as antibioticsor antimycotic compounds, including but not limited to, active agentssuch as antifungal agents, antibacterial agents, anti-viral agents andantiparasitic agents, and metals. An antimicrobial agent may comprise asubstance, compound or molecule, that kills or inhibits the growth ofmicroorganisms such as bacteria, fungi, or protozoans. Antimicrobialagents may either kill microbes (microbiocidal) or prevent the growth ofmicrobes (microbiostatic). Disinfectants are antimicrobial substancesused on non-living objects or outside the body. Anitmicrobial agentsinclude those obtained from natural sources, such as Beta-lactamantibiotics (such as penicillins, cephalosporins), and protein synthesisinhibitors (such as aminoglycosides, macrolides, tetracyclines,chloramphenicol, polypeptides), and those from synthetic sources such assulphonamides, cotrimoxazole, quinolones, anti-fungals, anti-cancerdrugs, antimalarials, anti-tuberculosis drugs, anti-leprotics, andanti-protozoals.

Examples of antimicrobial agents that can be used in the presentinvention include, but are not limited to, isoniazid, ethambutol,pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones,ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin,dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline,ainpicillin, amphotericin B, ketoconazole, fluconazole, pyrimethaniine,sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone,paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet,penicillin, gentamicin, ganciclovir, iatroconazole, miconazole,Zn-pyrithione, heavy metals including, but not limited to, gold,platinum, silver, zinc and copper, and their combined forms including,salts, such as chloride, bromide, iodide and periodate, and complexeswith carriers, and other forms. As used herein, the term metal includesall metal salts or metal compounds, including, but not limited to, metalchlorides, metal phosphates, metal sulfates, metal iodides or metalbromides. The active form of some metal salts is the ionic form. Otherantimicrobial agents include, but are not limited to, polyeneantifungals, Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin,Nystatin, Rimocidin, Imidazoles, Bifonazole, Butoconazole, Clotrimazole,Econazole, Fenticonazole, Isoconazole, Ketoconazole, Miconazole,Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole,Triazoles, Albaconazole, Fluconazole, Isavuconazole, Itraconazole,Posaconazole, Ravuconazole, Terconazole, Voriconazole, Thiazoles,Abafungin, Allylamines, Amorolfin, Butenafine, Naftifine, Terbinafine,chinocandins, Anidulafungin, Caspofungin, Micafungin.

Examples of microbial organisms that may be treated by the presentinvention include, but are not limited to, pathogenic fungi including,but not limited to, Candida species, Aspergillus species, Cryptococcusspecies, histoplasa species, stachybotrus species, and pneumacystusspecies; bacteria including but not limited to:

Acetobacter aurantius Calymmatobacterium granulomatis Acinetobacterbaumannii Campylobacter Actinomyces israelii Campylobacter coliAgrobacterium radiobacter Campylobacter fetus Agrobacterium tumefaciensCampylobacter jejuni Azorhizobium caulinodans Campylobacter pyloriAzotobacter vinelandii Chlamydia Anaplasma Chlamydia trachomatisAnaplasma phagocytophilum Chlamydophila Acetobacter aurantiusChlamydophila pneumoniae Bacillus Chlamydophila psittaci Bacillusanthracis Clostridium Bacillus brevis Clostridium botulinum Bacilluscereus Clostridium difficile Bacillus msiformis Clostridium perfringensBacillus liclieniformis Clostridium tetani Bacillus megateriumCorynebacterium Bacillus mycoides Corynebacterium diphtheriae Bacillusstearothermophilus Corynebacterium fusiforme Bacillus subtilis Coxiellaburnetii Bacteroides Ehrlichia chaffeensis Bacteroides fragilisEnterobacter cloacae Bacteroides gingivalis Enterococcus Bacteroidesmelaninogenicus Enterococcus avium Bartonella Enterococcus duransBartonella henselae Enterococcus faecalis Bartonella quintanaEnterococcus faecium Bordetella Enterococcus galllinarum Bordetellabronchiseptica Enterococcus maloratus Bordetella pertussis Escherichiacoli Borrelia burgdorferi Francisella tularensis Brucella Fusobacteriumnucleatum Brucella abortus Haemophilus Brucella melitensis Haemophilusducreyi Gardnerella vaginalis Haemophilus influenzae Klebsiellapneumoniae Haemophilus parainfluenzae Lactobacillus Haemophiluspertussis Lactobacillus acidophilus Haemophilus vaginalis Lactobacilluscasei Helicobacter pylori Lactococcus lactis Neisseria Legionellapneumophila Neisseria gonorrhoeae Listeria monocytogenes Neisseriameningitidis Lactobacillus Bulgaricus Rhizobium radiobacter MycoplasmaRickettsia Mycoplasma fermentans Rickettsia prowazekii Mycoplasmagenitalium Rickettsia psittaci Mycoplasma hominis Rickettsia quintanaMycoplasma penetrans Rickettsia rickettsii Mycoplasma pneumoniaeRickettsia trachomae Metlianobacterium extroquens RochalimaeaMicrobacterium multiforme Rochalimaea henselae Micrococcus luteusRochalimaea quintana Moraxella catarrhalis Rothia dentocariosaMycobacterium Rhizobium radiobacter Mycobacterium avium SalmonellaMycobacterium bovis Salmonella enteritidis Mycobacterium diphtheriaeSalmonella typhi Mycobacterium intracellulare Salmonella typhimuriumMycobacterium leprae Serratia marcescens Mycobacterium lepraemuriumShigella dysenteriae Mycobacterium phlei Staphylococcus Mycobacteriumsmegmatis Staphylococcus aureus Mycobacterium tuberculosisStaphylococcus epidermidis Pasteurella Stenotrophomonas maltophiliaPasteurella multocida Streptococcus Pasteurella tularensis Streptococcusagalactiae Peptostreptococcus Streptococcus avium Porphyromonasgingivalis Streptococcus bovis Pseudomonas aeruginosa Streptococcuscricetus Streptococcus mitior Streptococcus faceium Streptococcus mitisStreptococcus faecalis Streptococcus mutans Streptococcus ferusStreptococcus oralis Streptococcus gallinarum Streptococcus pneumoniaeStreptococcus lactis Streptococcus pyogenes Streptococcus sanguisStreptococcus rattus Streptococcus sobrinus Streptococcus salivariusWolbachia Treponema Vibrio parahaemolyticus Treponema pallidum Vibriovulnificus Treponema denticola Yersinia Vibrio Yersinia enterocoliticaVibrio cholerae Yersinia pestis Vibrio comma Yersinia pseudotuberculosis

The terms “treating”, “treatment”, “therapy”, and “therapeutictreatment” as used herein refer to curative therapy, prophylactictherapy, or preventative therapy. As used herein, the terms refers tothe medical management of a subject or a patient with the intent tocure, ameliorate, stabilize, or prevent a disease, pathologicalcondition, or disorder, such as, for example, cancer or a tumor. Thisterm includes active treatment, that is, treatment directed directedspecifically toward the improvement of a disease, pathologicalcondition, or disorder, disorder, and also includes causal treatment,that is, treatment directed toward removal of the cause of theassociated disease, pathological condition, or disorder. In addition,this term includes palliative treatment, that is, treatment designed forthe relief of symptoms rather than the curing of the disease,pathological condition, or disorder; preventative treatment, that is,treatment directed to minimizing or partially or completely inhibitingthe development of the associated disease, pathological condition, ordisorder; and supportive treatment, that is, treatment employed tosupplement another specific therapy directed toward the improvement ofthe associated disease, pathological condition, or disorder. In variousaspects, the term covers any treatment of a subject, including a mammal(e.g., a human), and includes: (i) preventing the disease from occurringin a subject that can be predisposed to the disease but has not yet beendiagnosed as having it; (ii) inhibiting the disease, i.e., arresting itsdevelopment; or (iii) relieving the disease, i.e., causing regression ofthe disease. In an aspect, the disease, pathological condition, ordisorder is cancer, such as, for example, breast cancer, lung cancer,colorectal, liver cancer, or pancreatic cancer.

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician or aresearcher, and found to have a condition that can be diagnosed ortreated by compositions or methods disclosed herein. For example,“diagnosed with cancer” means having been subjected to a physicalexamination by a person of skill, for example, a physician or aresearcher, and found to have a condition that can be diagnosed ortreated by a compound or composition that alleviates or amelioratescancer and/or aberrant cell growth.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., a disorder relatedto cancer and/or aberrant cell growth) based upon an earlier diagnosisby a person of skill and thereafter subjected to treatment for thedisorder. It is contemplated that the identification can, in one aspect,be performed by a person different from the person making the diagnosis.It is also contemplated, in a further aspect, that the administrationcan be performed by one who subsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a peptide (such as a CT20 Bax peptide), or acomposition (such as a composition comprising a CT20 Bax peptide), orpharmaceutical preparation (such as a preparation comprising a CT20ppeptide or a composition comprising a CT20 Bax peptide) to a subject.Such methods are well known to those skilled in the art and include, butare not limited to, intracardiac administration, oral administration,transdermal administration, administration by inhalation, nasaladministration, topical administration, intravaginal administration,ophthalmic administration, intraaural administration, intracerebraladministration, rectal administration, sublingual administration, buccaladministration, and parenteral administration, including injectable suchas intravenous administration, intraarterial administration,intramuscular administration, and subcutaneous administration.Administration can be continuous or intermittent. In various aspects, apreparation can be administered therapeutically; that is, administeredto treat an existing disease or condition. In further various aspects, apreparation can be administered prophylactically; that is, administeredfor prevention of a disease or condition.

The term “contacting” as used herein refers to bringing a disclosedcomposition or peptide or pharmaceutical preparation and a cell, targetreceptor, or other biological entity together in such a manner that thecompound can affect the activity of the target (e.g., receptor,transcription factor, cell, etc.), either directly; i.e., by interactingwith the target itself, or indirectly; i.e., by interacting with anothermolecule, co-factor, factor, or protein on which the activity of thetarget is dependent.

As used herein, the term “determining” can refer to measuring orascertaining a quantity or an amount or a change in expression and/oractivity level, e.g., of a nucleotide or transcript or polypeptide. Forexample, determining the amount of a disclosed transcript or polypeptidein a sample as used herein can refer to the steps that the skilledperson would take to measure or ascertain some quantifiable value of thetranscript or polypeptide in the sample. The art is familiar with theways to measure an amount of the disclosed nucleotides, transcripts,polypeptides, etc.

In an aspect, “determining” as used herein can refer to measuring orascertaining the level of cell death or cell survival, for example,following administration of a CT20p peptide or a composition comprisingan effective amount of a CT20p peptide. Methods of measuring orascertaining cell survival and cell death are known to the art andinclude, but are not limited to, histochemical staining (e.g., TUNEL),cell proliferation assay, cell death assays, morphological examination,etc. In an aspect, the size of a tumor can be measured non-invasivelythrough ultrasound.

As used herein, the term “level” refers to the amount of a targetmolecule in a sample, e.g., a sample from a subject. The amount of themolecule can be determined by any method known in the art and willdepend in part on the nature of the molecule (i.e., gene, mRNA, cDNA,protein, enzyme, etc.). The art is familiar with quantification methodsfor nucleotides (e.g., genes, cDNA, mRNA, etc) as well as proteins,polypeptides, enzymes, etc. It is understood that the amount or level ofa molecule in a sample need not be determined in absolute terms, but canbe determined in relative terms (e.g., when compare to a control or asham or an untreated sample).

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, in an aspect,an effective amount of a CT20p peptideis an amount that permeabilizescell membranes and/or kills cells without causing extraneous damage tosurrounding non-cancerous cells. For example, a “therapeuticallyeffective amount” refers to an amount that is sufficient to achieve thedesired therapeutic result or to have an effect on undesired symptoms,but is generally insufficient to cause adverse side affects. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration; the route of administration; therate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental coincidental withthe specific compound employed and like factors well known in themedical medical arts.

By “modulate” is meant to alter, by increase or decrease. As usedherein, a “modulator” can mean a composition that can either increase ordecrease the expression level or activity level of a gene or geneproduct such as a peptide. Modulation in expression or activity does nothave to be complete. For example, expression or activity can bemodulated by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, 100% or any percentage in between as compared to a control cellwherein the expression or activity of a gene or gene product has notbeen modulated by a composition.

As used herein, “EC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a CT20p peptide or a disclosed compositioncomprising a CT20p peptide) that is required for 50% enhancement oractivation of a biological process, or component of a process, includinga protein, subunit, organelle, ribonucleoprotein, etc. EC₅₀ also refersto the concentration or dose of a substance that is required for 50%enhancement or activation in vivo, as further defined elsewhere herein.Alternatively, EC₅₀ can refer to the concentration or dose of compoundthat provokes a response halfway between the baseline and maximumresponse. The response can be measured in an in vitro or in vivo systemas is convenient and appropriate for the biological response ofinterest. For example, the response can be measured in vitro usingcultured cancer cells or in an ex vivo organ culture system withisolated cancer cells, e.g., pancreatic cancer cells, breast cancercells, liver cancer cells, lung cancer cells, colorectal cancer cells,etc.). Alternatively, the response can be measured in vivo using anappropriate research model such as rodent, including mice and rats. Themouse or rat can be an inbred strain with phenotypic characteristics ofinterest such as, for example, cancer and/or aberrant cell growth. Asappropriate, the response can be measured in a transgenic or knockoutmouse or rat wherein a gene or genes has been introduced or knocked-out,as appropriate, to replicate a disease process.

As used herein, “IC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a CT20p peptide or a disclosed compositioncomprising a CT20p peptide) that is required for 50% inhibition ordiminution of a biological process, or component of a process, includinga protein, subunit, organelle, ribonucleoprotein, etc. IC₅₀ also refersto the concentration or dose of a substance that is required for 50%inhibition or diminution in vivo, as further defined elsewhere herein.Alternatively, IC₅₀ also refers to the half maximal (50%) inhibitoryconcentration (IC) or inhibitory dose of a substance. The response canbe measured in an in vitro or in vivo system as is convenient andappropriate for the biological response of interest. For example, theresponse can be measured in vitro using cultured cancer cells or in anex vivo organ culture system with isolated cancer cells (e.g., breastcancer cells, pancreatic cancer cells, liver cancer cells, lung cancercells, colorectal cancer cells, etc.). Alternatively, the response canbe measured in vivo using an appropriate research model such as rodent,including mice and rats. The mouse or rat can be an inbred strain withphenotypic characteristics of interest such as, for example, cancerand/or aberrant cell growth. As appropriate, the response can bemeasured in a transgenic or knockout mouse or rat wherein a gene orgenes has been introduced or knocked-out, as appropriate, to replicate adisease process.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner. As used herein, the term “pharmaceuticallyacceptable carrier” refers to sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol and the like),carboxymethylcellulose and suitable mixtures thereof, vegetable oils(such as olive oil) and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials materials such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions and by the use of surfactants.These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents such as paraben, chlorobutanol,phenol, sorbic acid and the like. It can also be desirable to includeisotonic agents such as sugars, sodium chloride and the like. ProlongedProlonged absorption of the injectable pharmaceutical form can bebrought about by the inclusion of agents, such as aluminum monostearateand gelatin, which delay absorption. Injectable depot forms are made byforming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

As used herein, the term “cancer” refers to a proliferative disorder ordisease caused or characterized by the proliferation of cells which havelost susceptibility to normal growth control. The term “cancer” includestumors and any other proliferative disorders. Cancers of the same tissuetype originate in the same tissue, and may be divided into differentsubtypes based on their biological characteristics. Cancer includes, butis not limited to, melanoma, leukemia, astocytoma, glioblastoma,lymphoma, glioma, Hodgkins lymphoma, and chronic lymphocyte leukemia.Cancer also includes, but is not limited to, cancer of the brain, bone,pancreas, lung, liver, breast, thyroid, ovary, uterus, testis,pituitary, kidney, stomach, esophagus, anus, and rectum.

As used herein, the term “sensitizing” refers to an increasedsensitivity of a cell or a subject to a treatment, such as a therapeutictreatment. The term “sensitizing” also refers to a reduction or decreasein the resistance of a cancer cell or a subject with cancer inresponding to a therapeutic treatment. An increased sensitivity or areduced sensitivity to a therapeutic treatment is measured according toa known method in the art for the particular treatment and methodsincluding, but not limited to, cell proliferation assays and cell deathassays. The sensitivity or resistance may also be measured in a subjectby measuring the tumor size reduction over a period of time, such as,for example, every 1 to 3 to 6 month for a human subject and every 2 to4 to 6 weeks for non-human subject (e.g., mouse or rat). The sensitivityof a cell or a subject to treatment can be measured or determined bycomparing the sensitivity of a cell or a subject followingadministration of a CT20p peptide or a composition comprising aneffective amount of a CT20p peptide to the sensitivity of a cell orsubject that has not been administered a CT20p peptide or a compositioncomprising an effective amount of a CT20p peptide.

As used herein, the term “anti-cancer” or “anti-neoplastic” drug refersto one or more drugs that can be used in conjunction with a CT20ppeptide or a composition comprising an effective amount of a CT20ppeptide to treat cancer and/or aberrant cell growth. Examples ofanti-cancer drugs or anti-neoplastic drugs include, but are not limitedto, the following: Acivicin; Aclarubicin; Acodazole Hydrochloride;AcrQnine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; AmetantroneAcetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin;Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat;Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate;Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; DaunorubicinHydrochloride; Decitabine; Dexormaplatin; Dezaguanine; DezaguanineMesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate;Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone;Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine;Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; InterferonAlfa-n3; Interferon Beta-1a; Interferon Gamma 1 b; Iproplatin;Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; LeuprolideAcetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine;Losoxantrone Hydrochloride; Masoprocol; Maytansine; MechlorethamineHydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; MycophenolicAcid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid;Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin;Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine;Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; ToremifeneCitrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate;Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate;Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate;Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin;Zinostatin; Zorubicin Hydrochloride.

Other anti-neoplastic compounds include: 20-epi-1,25 dihydroxyvitaminD3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; atrsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, antiandrogen, prostatic carcinoma;antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolinglycinate; apoptosis gene modulators; apoptosis regulators; apurinicacid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin;diphenyl spiromustine; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocannycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; 4-ipomeanol; irinotecan;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance genieinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone +pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives;palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfatesodium; pentostatin; pentrozole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetinA; placetin B; plasminogen activator inhibitor;platinum complex;platinum compounds; platinum-triamine complex; porfimer sodium;porfiromycin; propyl bis-acridone; prostaglandin J2; proteasomeinhibitors; protein A-based immune modulator; protein kinase Cinhibitors; microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B 1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfmosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene dichloride; topotecan; topsentin; toremifene; totipotent stemcell factor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; velaresol; veramine; verdins; verteporfin; vinorelbine;vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;zinostatin stimalamer.

As used herein, radiosensitizers make a cancer cell more likely to bedamaged. Radiosensitizers enhance the sensitivity of cancer cells and/ora tumor to ionizing radiation, thereby increasing the efficacy ofradiotherapy. Examples of radiosensitizers include gemcitabine,5-fluorouracil, pentoxifylline, and vinorelbine.

The majority of chemotherapeutic drugs can be divided in to: alkylatingagents (e.g., cisplatin, carboplatin, oxaliplatin, mechloethamine,cyclophosphamide, chlorambucil, anti-metabolites (e.g., azathioprine,mercaptopurine), anthracyclines, plant alkaloids and terpenoids (e.g.,vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine,and podophyllotoxin) and taxanes (e.g., paclitaxel and docetaxel),topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine,etoposide, etoposide phosphate, and teniposide), monoclonal antibodies(e.g., trastuzumab, cetuximab, rituximab, bevacizumab), other antitumouragents (e.g., dactinomycin), and hormonal therapy (e.g., steroids suchas dexamethasone), finasteride, aromatase inhibitors, andgonadotropin-releasing hormone agonists).

Disclosed are the components to be used to prepare a composition of theinvention as well as the compositions themselves to be used within themethods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds can not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds compounds are discussed, specificallycontemplated is each and every combination and permutation of thecompound and the modifications that are possible unless specificallyindicated to the contrary. Thus, if a class of molecules A, B, and C aredisclosed as well as a class of molecules D, E, and F and an example ofa combination molecule, A-D is disclosed, then even if each is notindividually recited each is individually and collectively contemplatedcontemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E,and C-F are considered disclosed. Likewise, any subset or combination ofthese is also disclosed. Thus, for example, the sub-group of A-E, B-F,and C-E would be considered disclosed. This concept applies to allaspects of this application including, but not limited to, steps inmethods methods of making and using the compositions of the invention.Thus, if there are a variety of of additional steps that can beperformed it is understood that each of these additional steps can beperformed with any specific embodiment or combination of embodiments ofthe methods of the invention.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The invention illustratively described herein suitablycan be practiced in the absence of any element or elements, limitationor limitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of, and “consisting of can be replaced witheither of the other two terms, while retaining their ordinary meanings.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by embodiments,optional features, modification and variation of the concepts hereindisclosed can be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the description and the appended claims.

D. Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric.

1. General Experiments a. Establishment of Cell Lines

The Flp-In T-REx-293 cell line (Invitrogen) stably expressed thelacZ-Zeocin fusion gene and Tet repressor. The 293 line was maintainedin Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS)(tetracyc line-reduced), 2 mM L-glutamine and 1%Penicillin-Streptomycin. The HCT-116 Bax^(−/−) and Bax^(+/+) colorectalcancer cell lines (Zhang et al., 2000) were maintained in McCoy's 5Amedia, 10% FBS, and 1% Penicillin-Streptomycin. The breast cancer celllines, MCF-7 and MCB-MD-231 (ATCC), were maintained in DMEM, 10% FBS,and 1% Penicillin-Streptomycin. MCF-7 cells were supplemented with 1%L-Glutamine every 15 days. Early passages of all cell lines were frozenas stocks at time of receipt. Cell lines were used at less than 10passages from stocks.

b. Generation of the CT20 Bax Peptide

The CT20p peptide (Ac-VITFVAGVLTASLTIWKKMG-NH2) (Biopeptide Co., Inc.)(SEQ ID NO:53) was commercially synthesized at >98% purity.

c. Generation of Plasmids for Mutagenesis and Transfection

For inducible expression of Bax, the Flp-In T-REx System (Invitrogen)was utilized according to the manufacturer's protocol. Briefly,PCR-directed mutagenesis of K189/K190 was performed using HA-taggedprimer sets (Table 1). Bax constructs were amplified from pEGFP-Bax,digested with EcoRV, and cloned into the plasmid pcDNA5/FRT/TO, whichundergoes DNA recombination at the Flp Recombination Target (FRT) sitewhen co-expressed with the Flp recombinase pOG44 plasmid. Constructswere confirmed by sequencing. Fugene transfection reagent (Roche) wasused to co-transfect plasmids at a ratio of 9:1. Stable Flp-In T-RExexpression cell lines were selected for Blasticidin resistance (10μg/mL), Hygromycin resistance (100 μg/mL) and Zeocin sensitivity (200μg/mL). Bax expression was induced with 1 μg/mL tetracycline. Cells wereassayed after 24 hours of induction. See Table 1 for sequences andprimers used herein.

To generate the Destabilization Domain (DD)-tagged Bax CT (amino acids173-192) fusion proteins with K189/K190 (wild-type) or EE, LL, and RRmutations, primers (Table 1) were annealed and ligated into theProteoTuner vector (Clontech) digested by EcoRI and BamHI. Generation ofDD-tagged, full-length WT Bax was previously described (Boohaker et al.,2011). The ProteoTuner IRES2 system (Clontech) also had the markerprotein GFP downstream to the internal ribosome entry sequence (IRES)and was translated independently of the DD-tagged protein. Cells weretransiently transfected using the TransIT-LT1 transfection reagent(Minis) for 24 hours and microscopically assayed for GFP expression.Expression of DD-tagged proteins was induced for 4-5 hours by adding 500nM of Shield (Clontech).

MDA-MB-231 cells were transiently transfected with pcDNA-Bcl2 (or ascontrol pEGFP (Clontech)) using the TransIT-LT1 transfection reagent(Minis). To assess transfection efficiency, cells were assayedmicroscopically for EGFP expression. To assess Bcl-2 expression, cellslysates were immunoblotted.

d. Detection of Mitochondrial Translocation

Mitochondrial and cytosolic proteins were isolated using a mitochondrialenrichment kit (Pierce). Western blots were run using 12-15% SDS-PAGEgels and PVDF membranes and probed with the following primaryantibodies: 16B12 anti-HA mouse monoclonal (Covance) for HA-tagged Bax;631073Anti-DD monoclonal (Clontech) for DD-Bax; N-20 (Santa Cruz) forendogenous Bax; Ab-2 (Fitzgerald) for prohibitin; C20 (MAPK) (SantaCruz) for p38 MAP kinase; and rabbit polyclonal for Bcl-2 (Santa Cruz).The appropriate secondary antibodies conjugated to horseradishperoxidase (HRP) was then used and visualized with enhancedchemiluminescence kit (Pierce). Molecular weight markers (SeeBlue Plus 2(Invitrogen)) were used to approximate the position of protein bands inblots.

e. Co-Localization of Mitochondrial Proteins

To determine whether expression of DD-tagged Bax C-terminal peptidesco-localized with mitochondria, Bax^(+/+) HCT-116 cells and Bax^(−/−)HCT-116 cells were cultured for 24 hours on coverslips pretreated withlaminin. The DD-Bax C-terminal WT peptide and DD-Bax C-terminal EE, LL,and RR mutations constructs were transfected into the cells using MinisLT-1 reagent according manufacturer's protocol. After 24 hours,expression of the transfected peptides was induced by the addition ofshield 1 for 4 hrs. Cells were fixed with 2% w/v formaldehyde/PBS for 15minutes and permeabilized using 0.05% Triton X-100/PBS for 15 minutes.After washing, cells were incubated with primary antibodies HSP60(H-300, Santa Cruz) and DD monoclonal antibody for 1 hour at roomtemperature, which was followed by incubation with secondaryanti-rabbit-Cy3 (81-6115, Invitrogen) and anti-mouse-Texas red(715076020, Jackson Immunoresearch) for 30 minutes. After the finalwash, cells were mounted with gel/mount medium (Mol, Biomeda) and imageswere acquired with UltraView (PerkinElmer) microscopy with aplan-apochromat 63×/1.4 oil objective. The scanned images were processedand Pearson's correlation coefficients determined using Velocity Version5.5 (Perkin Elmer).

f. Generation and Expression of EGFP-CT20p Peptide Fusion Constructs

To generate the EGFP-CT20p peptide fusion proteins, primersincorporating the CT (amino acids 173-192) of Bax with mutationstargeting K189/K190 (Table 1) were used to amplify EGFP from thetemplate pEGFP (Clontech). The PCR insert was cloned into pcDNA5/FRT/TOas previously above. HCT-116 cells were transiently transfected usingthe TransIT-LT1 transfection reagent (Minis) and cells assayedmicroscopically for EGFP expression up to 12 hours later.

g. Confocal Imaging of Live Cells

Images were acquired through a PerkinElmer UltraView spinning discconfocal system with AxioObserver.Z1 stand (Carl Zeiss) in a humidityand temperature-controlled chamber (LiveCell) with cells cultured onMatTek plates (MatTek Corporation). Post-acquisition snapshots weretaken from time-lapse movies at time points indicated in the figures.For EGFP-CT20 Bax fusion proteins, time-lapse movies were initiated twohours after transfection and images acquired through 12 hours ofexpression using a Plan-Apochromat 10× objective. For DD-CT20 Bax fusionproteins, cells were incubated with 1 nM MitoTracker Red 580 for 30minutes prior to imaging. Time-lapse movies were recorded for up to 12hours using a Plan-Apochromat 63× Oil DIC objective.

Visualization of the uptake and effects of the fluorescent dye(DiI)-loaded nanoparticles in HCT-116, MCF-7, and MDA-MD-231 cells wasobserved using a 10× air objective with a numerical aperture of 0.3,using ex/em of 514/587. All cells were loaded with MitoTracker.Visualization of the HCT-116 cell lines was observed usingPlan-Apochromat 63× Oil objective. MCF-7 and MDA-MB-231 images werecaptured using Plan-Apochromat 40× Oil objective. All time-lapse imageswere generated in 2D by capturing 6 time points per hour for 24 hours ofthe same field.

h. Treatments and Detection of Apoptotic Cells by Flow Cytometry

HCT-116, MCF-7, or MDA-MB-231 cells were collected at a finalconcentration of 1×10⁶ cells/mL and assayed using the SYTOX® AADvanced™dead cell stain solution (Invitrogen). Cells were analyzed using the BDFACSCanto flow cytometer. SYTOX® AADvanced™ was visualized at 488 nm andemissions collected at 695 nm. Analysis of data was done using FSCExpress software (DeNovo). Membrane asymmetry was assessed using theViolet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit(Invitrogen) according to the manufacturer's protocol.

To evaluate apoptosis, cells were pre-treated with 100 μM of thepan-caspase inhibitor Z-VAD-fmk (EMD Biosciences) or transientlytransfected with Bcl-2, then treated with either CT20/nanoparticles orcisplatin (CDDP) alone or in combination as indicated in figure legends.Following treatment, cells were analyzed for cell death and membraneasymmetry.

i. Synthesis of Polymeric Nanoparticles Encapsulating the Bax CT20 BaxPeptide

The Bax CT20p peptidewas encapsulated into hyperbranched polymeric(HBPE) nanoparticles following a previously reported method (Santra etal., 2010). A fluorescent dye (DiI) was co-encapsulated with thepeptide. In brief, 1.0 μL, of DiI dye (10 μg/μL) and 36 μL, of CT20ppeptide (0.05 μg/μL) solution in 250 μL, of DMSO were mixed in 250 μL ofa DMSO solution containing the HBPE polymer (12 mg) for a ratio of ˜0.15μg peptide: 1 mg nanoparticles. The resulting polymer-DiI/Bax mixture inDMSO was added to deionized water (2.5 mL) to form the HBPE (BaxCT20/DiI) nanoparticles. The resulting nanoparticles were purified usinga PD-10 column and dialyzed (MWCO 6-8K) against PBS (pH=7.4). Dynamiclight scattering and zeta potential analysis of the nanoparticle revealsa size diameter of 88±2 nm and zeta potential of −54.5 mV.

j. Synthesis of Aminated Polymeric Nanoparticles Encapsulating the CT20Bax Peptide

The HBPE nanoparticles contain functional carboxylic groups on theirsurface that resulted in a negative charge. To introduce a positivelycharged surface, the nanoparticles were aminated using water-solublecarbodiimide chemistry [EDC: 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride and NHS: N-hydroxysuccinimide chemistry],following a previously reported method (Santra et al., 2010). Briefly,to a solution of HBPE (Bax CT20/DiI) nanoparticles (1.0 mmol) in PBS(pH=7.4), a solution of EDC (10 mmol) and NHS (10 mmol) in MES buffer(pH=6.0) was added. Afterwards, ethylenediamine (10 mmol) in DMSO wasadded to obtain aminated DiI/Bax co-encapsulation polymericnanoparticles (PNPs), which were purified and dialyzed. Dynamic lightscattering and zeta potential analysis of the nanoparticle reveals ananoparticle size diameter or 91±3 nm and zeta potential of +10.3 mV.All nanoparticles were stored at 4° C. A final working concentration of350 pM was determined by testing the toxicity of 7 nM, 1.4 nM, 0.7 nM,and 0.35 nM using HCT-116^(+/+) cells.

k. Determination of Calcein Release

Calcein release from artificial membranes was measured on a JASCO 810spectropolarimeter (Jasco Inc.) with a Peltier water cooled thermostatand a photomultiplier tube mounted at 90 degrees for fluorescencemeasurements. Large unilamellar vesicles (LUVs) were prepared with thefollowing modifications: Lipids (Avanti Polar Lipids) in chloroform weremixed in the following molar ratios: 52.5%1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC), 21%1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylethanolamine (POPE), 13%bovine liver L-α phosphatidylinositol (PI), 10% cholesterol and 3.5%1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylglycerol (POPG). Theserations mimicked the outer mitochondrial membrane. After removingchloroform and desiccating, the dried lipid film was resuspended in 50mM HEPES, pH 7, supplemented with 110 mM NaCl and 80 mM calcein andextruded with Avanti's mini extruder (Alabaster, Ala.). External calceinwas removed by gel filtration through a 1.5×50 cm Econo-Column (Bio-Rad)freshly packed with Sephadex 50 (GE Healthcare). CT20 Bax peptides wereadded at concentrations equivalent to those used with cells in culture.Calcein fluorescence was excited at 495 nm and emission spectra recordedbetween 510 and 550 nm (excitation/ emission slits: 10/3 nm). Sampleswere maintained at 37° C. and a final measurement taken at 24 hours.Maximum calcein release was obtained by the addition of Triton ×100(0.1% final concentration) to calcein loaded LUVs without nanoparticlesthat had had been incubated at 37° C. for 24 hours after the addition of10 μL DMSO.

1. In Vivo Experiments

One to two million MBA-MD-231 cells were harvested from culture andinjected subcutaneously into the right and left flanks of 16 female nudemice (nu/nu, Charles River). After 2-3 weeks, tumor volume and growthwas assessed by ultrasound (VisualSonics Vevo 2100). Mice with tumorswere injected intratumorally with PBS, unloaded polymeric nanoparticles,or CT20 Bax peptide-loaded polymeric nanoparticles at 4× theconcentrations described above. Injections were performed twice over a4-5 day period. Mice were observed 0-5 days post-treatments and tumorvolume assessed by ultrasound.

2. C-Terminal α9 Helix of Bax Enables Membrane Binding andPermeabilization

To develop a novel anti-cancer agent that could be used to sensitizecancer cells to treatment, the death-inducing activity of Bax wasexamined. The protein's C-terminal domain was a point of focus becauseof its putative membrane-binding properties (Ausili et al., 2008;Martinez-Senac et al., 2001; Nechushtan et al., 1999; Schinzel et al.,2004). Mutagenesis of the C-terminus of full-length Bax was performed(Table 2). As the constitutive expression of N-terminal-tagged Baxinduced spontaneous cell death. To avoid this, full-length HA-tagged Baxwas expressed in stably transfected Flp-In T-REx 293 cells. Baxconstructs were integrated into the genome at a single FRT(recombination) site and the levels of Bax expression in these isogeniccell lines did not cause apoptosis. These experiments determined therole of the C-terminus and amino acids K189/K190 in the localization ofBax under normal cellular conditions.

TABLE 2 Effect of K189/K190 Mutations on Bax Intracellular Localization.Residues (189-190-191- Cellular Bax C-terminus 192) SEQ ID NOLocalization Wild Type KKMG 54 Cyto > Mito (+) to hydrophobic LKMG 55Mito KLMG 56 LLMG 57 (+) to (−) charge EKMG 58 Cytosol reversal KEMK 59EEMG 60 (+) to polar QQMG 61 Cyto = Mito (+) to (−) charge DDMG 62Cytosol reversal Increase (+) charge, RRMG 63 Mito length Rearrangementof KMGK 64 Cyto = Mito (++) Deletion of (+) -KMG Cyto = Mito Deletion of(+) and -MGK Cytosol rearrangement

Localization of full-length, wild-type (WT) Bax (Bax-KK) was distributedamong cytosolic and mitochondrial lysates (FIG. 1A). Specifically,mitochondrial translocation of HA-tagged wild type Bax (Bax-KK) andK189/K190 mutants, expressed in 293 cells using the Flp-In T-Rex system,was examined by immunoblot. p38 MAPK and prohibitin were blotted forcytosolic and mitochondrial content, respectively. Data arerepresentative of five independent assays. Images from full-length blotswere cropped for concise presentation. N-terminal-deleted Bax (Bax-ΔNT)was localized primarily to mitochondria while the C-terminal-deleted Bax(Bax-ΔCT) was retained in the cytosol. These data indicate the role ofthe N- and C-terminal domains in Bax localization (FIG. 1A).

Substitution of K189/K190 with negatively charged residues, asparticacid (D) or glutamic acid (E), resulted in the cytosolic retention ofBax (Bax-DD, Bax-EE) (FIG. 1B). Substitution of K189/K190 withpositively charged arginine (R) (Bax-RR) led to mitochondriallocalization (FIG. 1B). Substitution of K189/K190 with a polar aminoacid, glutamine (Q) (Bax-QQ), led to less mitochondrial Bax, whencompared Bax-KK and Bax-RR (FIGS. 1A and 1B). Substitution of K189/K190with the hydrophobic amino acid leucine (L) (Bax-LL) resulted inmitochondrial association (FIG. 1A). Mutation of K189 (Bax-EK) alsorendered Bax cytosolic, which was not observed by mutation of K190(Bax-KMGK) (FIGS. 1A and 1B, Table 2). These results demonstrated thatthe C-terminus of Bax, and K189/K190, modulated the association of thefull-length protein with mitochondria.

Analysis of the effects of mutations of K189/K190 in full-length Baxindicated that the C-terminal domain was a preferred region from whichto fashion a membrane-binding and possibly a membrane-permeabilizingpeptide. Using a commercially synthesized peptide composed of the lasttwenty amino acids (172-192) of the C-terminus of Bax (CT20), theability of CT20p peptideto insert and permeabilize lipid membranes wasexamined. Liposomes contained phospholipids that composed themitochondrial outer membrane and were loaded with calcein. FIG. 1Cdemonstrates that the CT20p peptideinserted itself intomitochondrial-like lipid vesicles and caused the release of calcein. InFIG. 1C, the red lines indicate maximal release of calcein with TritonX-100. The blue lines indicate treatment with DMSO and the green lines(except for control) indicate treatment with CT20 Bax peptides. A mutantCT20 Bax peptide, in which the terminal lysines (K189/K190 in thefull-length protein) were exchanged with leucine (LL) or aspartic acid(EE), did not cause calcein release. This indicated that the originallysines at positions 189 and 190 were required for optimal membranepermeabilization. CT20 Bax or mutant CT20-LL peptides stabilized thelipid membranes and impaired the maximal release of calcein upontreatment with detergent (FIG. 1C). However, this did not occur with theCT20-EE peptide, indicating that the amino acid sequence of the peptidedictated the nature of the interaction with lipid membranes. These datademonstrated that the CT20 peptide bound to lipid membranes and causedthe release of vesicular contents, and further demonstrated that theCT20p peptideadded rigidity or structure to the lipid membranes.

3. Expression of the CT20p Peptide Induces Cell Death

Having established the importance of the C-terminal domain of Bax in theintracellular localization of the protein and demonstrated themembrane-binding and permeabilization properties of the CT20 Baxpeptide, the capacity of the CT20p peptide to induce cell death was nextevaluated. Specifically, whether the CT20p peptide had cytotoxicproperties based on its capacity to associate with lipid membranes oforganelles like mitochondria was assessed. The CT20p peptide domain wasfused to a short destabilization domain (DD) for detection and inducibleexpression. The CT20p peptide fused to DD (DD-CT20) was induciblyexpressed in HCT-116 cells and peptide localization and induction ofcell death was examined. For a control, the action of the DD-CT20 (KKand EE, LL and RR mutants) peptides to DD-tagged full-length (FL) Baxwas compared. Induced expression of DD-FL Bax did not cause cell deathin the absence of apoptotic stimuli.

Gene expression was induced for four hours after previous transfectionof cells with DD-FL Bax or DD-CT20 peptide constructs. Constitutive GFPexpression on the bi-cistronic plasmid was controlled by an IRES elementand used to detect transfected cells. The mitochondrial translocation ofDD-tagged Bax full-length (FL-Bax) and DD-tagged CT20 peptides,wild-type and EE, LL and RR mutants, was examined in Bax^(+/+) HCT-116cells by immunoblot. As shown in FIG. 2A, most of the DD-tagged FL Baxwas found in cytosolic extracts. A fraction of DD-FL-Bax was also foundin mitochondria extracts. The small 15-16 kD band of DD-CT20 KK peptidesor DD-CT20 mutant peptides (EE, LL, and RR (fainter band)) was detectedin mitochondrial extracts, indicating that the DD-CT20 peptides weretargeting mitochondria.

Expression of DD-CT20 Bax also caused the mitochondrial translocation ofa small amount endogenous Bax (FIG. 2A). Data are representative of twoindependent assays. In FIG. 2A, endogenous Bax was probed with anti-Baxantibody. p38 MAPK and prohibitin indicated cytosolic and mitochondrialcontent, respectively. DD-fusions were detected with an anti-DDantibody. Controls were cells transfected with empty vector oruntransfected. Images from full-length blots were cropped for concisepresentation.

These results were confirmed by immunofluorescence (FIG. 7). In FIG. 7A,cells were fixed and double-stained with primary antibodies for HSP60and DD. The fixed cells were then incubated with secondaryanti-rabbit-Cy3 and anti-mouse-Texas red. The overlay appears as white.In FIG. 7B, the bar graphs indicate the quantitative assay ofco-localization of DD-tagged peptides to mitochondria. The measurementsare represented by three samples.

Experiments attaching the CT20 peptides to EGFP confirmed the findingsthat fusion of the CT20p peptide could confer membrane bindingproperties (FIGS. 8A and 8B). In FIGS. 8A and 8B, the mitochondrialtranslocation of EGFP-tagged with Bax CT (EGFP-KK) and K189/K190 mutantswas examined in Bax^(+/+) HCT-116 cells (A) and Bax^(−/−) HCT-116 cells(B) by immunoblot. p38 MAPK and Prohibitin were assessed for cytosolicand mitochondrial content, respectively. Control sample areuntransfected cells. Data are representative of five independent assays.Images from full-length blots were cropped for concise presentation.

In FIGS. 8C and 8D, Bax^(+/+) HCT-116 cells and Bax^(−/−) HCT-116 cells,respectively, were transfected with the EGFP-KK or K189/K190 mutants.Time-lapse movies were acquired with a 63× Oil objective. For eachsample, three different fields of view were acquired. Images shown aresnapshots taken at specified time points.

The toxicity of DD-CT20 KK peptide was shown when expression was inducedin HCT-116 cells (FIG. 2B for Bax^(+/+) cells and FIG. 2C for Bax^(−/−)cells). For each sample, three different fields of view were acquired.Images are representative “snapshots” of four independent experiments.Within five hours of gene induction, the DD-CT20 KK peptide causedmembrane perturbations and the death of Bax-containing cells. Similarresults were observed with the other forms of DD-CT20 (EE, LL and RR)peptides, although the death kinetics varied depending on the mutation(LL and RR mutants took longer to cause death) (FIG. 2B, 5 hours; insetsin FIG. 2B were increased 3-fold). Moreover, Bax deficiency did notimpair the lethality of the CT20 Bax peptide. This was observed byexpressing DD-CT20 KK in Bax^(−/−) HCT 116 cells, which caused celldeath detectable by 3-5 hours (FIG. 2C, 12 hours). DD-CT20 EE causedmembrane disturbances by 1 hour of induction and death by 3 hours. Thiswas followed by DD-CT20 RR, with loss of membrane integrity observedwithin 3 hours, and DD-CT20 LL causing membrane fluctuations by 5 hoursand death by 11 or 12 hours (FIG. 2C). While mutation of the C-terminallysines did not block mitochondrial translocation (FIG. 2A) or preventcell death, it did alter the timeframe in which death occurred. Thesefinding were confirmed with multiple similar experiments, whichaccounted for differences in transfection efficiency and expression ofindividual constructs. The data indicate that the C-terminal lysineswere needed to “fine tune” the interaction of the CT20 peptide withmitochondrial membranes, altering its capacity to induce cell death.(See FIG. 1C showing that CT20 peptides (WT and mutants) associated withlipid membranes, which affected membrane rigidity, but that only theCT20 WT (containing K189/K190) enabled the maximal release of calceinfrom loaded vesicles).

4. Delivery of the CT20 Peptide Using Polymeric Nanoparticles KillsCancer Cells

To determine whether the CT20p peptide could directly kill cancer cells,the CT20 wild type peptide was encapsulated in polymeric nanoparticles.As the CT20p peptide is amphipathic, it can be encapsulated within thehydrophobic pockets of aliphatic polymeric nanoparticles. FIG. 3A showsa schematic representation of the three dimensional structure ofaliphatic hyperbranched nanoparticles, in which DiI (fluorescent dye)and commercially synthesized CT20 Bax peptides were encapsulated intopositively charged, aminated (AM) or negatively charged, carboxylated(COOH) nanoparticles (Santra et al., 2010). To verify that thenanoparticles would not release the CT20 peptide at neutral pH, calceinloaded liposomes were prepared. While the CT20p peptide alone (350 pM)did induce the release of calcein (as shown in FIG. 1C), nanoparticlesloaded with CT20 peptide (350 pM) did not induce the release of calceinfrom liposomes, indicating that the nanoparticles were intact at pH 7(FIG. 9).

In FIG. 9, a calcein release assay was performed with calcein loadedlipid vesicles prepared with the composition of the mitochondrialmembrane. Calcein release was measured as fluorescence. Controls werecalcein release achieved with CT20p peptide alone and maximum calceinrelease obtained upon addition of Triton x100. A representativeexperiment of two independent experiments is shown. This confirmed thatcargo is released from nanoparticles only by intracellular esterases oracidic pH (Santra et al., 2010).

The uptake of DiI-loaded nanoparticles and effect upon the viability ofHCT-116 cells was evaluated. HCT-116 cells were treated withnanoparticles loaded with DiI or DiI+CT20 peptide (0.07 nM) for 24hours. FIG. 3B shows that HCT-116 cells took up nanoparticles, more sofor the Bax-deficient cells that were highly glycolytic (Boohaker etal., 2011). While treatment of HCT-116 cells with nanoparticles(unloaded) did not cause significant cell death (FIG. 3B), treatment ofHCT-116 cells with CT20 peptide-loaded nanoparticles at a concentrationof 700 pM (or 0.07 nM), caused rapid cell death (FIG. 3B). For eachsample, three different fields of view were acquired. Images arerepresentative “snapshots” of two independent experiments.

To demonstrate that nanoparticles alone (i.e., without the CT20 peptide)did not cause death, a DNA-binding dye (Sytox) was utilized to detectmembrane rupture of dead cells. FIG. 3C shows that minimal (Bax^(+/+)HCT-116) to no (Bax^(−/−) HCT-116) cell death was detected upon additionof DiI-loaded nanoparticles (5 μg, 10 μg or 15 μg) as compared to thepositive control (dead cells). To minimize off-target effects, allsubsequent experiments were performed at the lowest dose of the CT20peptide (350 pM)-nanoparticles (2.5 μg).

To visualize the effect of CT20 Bax peptide-nanoparticles uponmitochondria, Bax-containing or Bax-deficient HCT-116 cells were stainedwith Mitotracker and imaged live cells. Bax^(+/+) (FIG. 4A) andBax^(−/−) (FIG. 4B) HCT-116 cells were treated with AM- orCOOH-nanoparticles loaded with CT20 peptide (350 pM) for 24 hours. Tovisualize mitochondria, cells were treated with MitoTracker Red 580 andtime-lapse movies were acquired using a 63× Oil objective. FIGS. 4A and4B show “snapshots” at 0, 12, and 24 hour time points. These snapshotsrevealed changes in cell morphology, which changes included disruptionof mitochondria (reduced or faint Mitotracker staining), as well as cellshrinkage and membrane perturbations, which were indicative of celldeath.

Similarly, HCT-116 cells were treated with nanoparticles loaded withCT20 peptide (350 pM). Cell death was measured and the loss of membraneintegrity was detected within three hours of treatment with CT20 Baxpeptide-nanoparticles (FIG. 4C). Both AM-nanoparticles andCOOH-nanoparticles containing CT20p peptide were efficacious ininitiating cell death (FIG. 4C), although the COOH-nanoparticleformulation appeared more effective. These results coupled with those inFIG. 2 indicated that the death-inducing activity of the CT20p peptidewas independent of endogenous Bax. With respect to FIGS. 4A-C, threedifferent fields of view were acquired for each sample. Images arerepresentative “snapshots” of three independent experiments.

Whether the CT20 peptide was able to kill the breast cancer cells, MCF-7and MDA-MB-231, was examined. As shown in FIG. 5A, morphological changessuch as cell shrinkage and membrane perturbations in MCF-7 cellsfollowing treatment for 24 hours with AM- or COOH-nanoparticlescontaining the CT20p peptide(350 pM) were observed. For each sample,three different fields of view were acquired. Images are representative“snapshots” of three independent experiments Most MCF-7 cells diedwithin 24 hours, while loss of membrane integrity was detected by 3hours of treatment (FIG. 5B). In FIG. 5C, the live cell imagingexperiment revealed vacuolization, membrane fluctuations, and cellshrinkage of MDA-MB-231 cells following treatment with CT20peptide-nanoparticles. Within three hours, increased membrane rupturewas detected in MDA-MB-231 cells treated with COOH-nanoparticles loadedwith CT20 peptide (FIG. 5D). These results demonstrated that the CT20peptide, once introduced into cells, rapidly triggered cell death. Whilethe CT20 peptide encapsulated in nanoparticles caused the death of fourcancer cell lines (colon and breast), the CT20 Bax peptide/nanoparticleformulation was less effective inducing death in two lung cancer celllines. This data indicated that that differences in the uptake andefficacy of the CT20 peptide/nanoparticles could exist that are relatedto the physiology of the cancer cell.

5. The Death-Inducing Activity of the CT20p Peptide is Independent ofCaspases and Resistant to BCL-2.

To investigate the mechanism by which the CT20p peptide was inducingcell death, MDA-MB-231 cells were treated with CT20 Baxpeptide/nanoparticles (350 pM). A caspase inhibitor (e.g., ZVAD-Fmkand/or CDDP) was added or Bcl-2 was overexpressed. As shown in FIG. 6A,the CT20 peptide caused significant loss of membrane integrity, whichwas largely unaffected by caspase inhibition with Z-VAD-FMK. Incontrast, treatment of MDA-MB-231 cells with cisplatin (CDDP) inducedcell death that was inhibited by ZVAD-FMK. These results indicated thatthe death pathway induced by the CT20p peptide was different than CDDPand was independent of the effector caspases inhibited by ZVAD-FMK. WhenBcl-2 was overexpressed, the overexpression did not impair the deathactivity of the CT20 Bax peptide, but rather slightly enhanced it. Bcl-2overexpression impaired death induced by CDDP (FIG. 6A). Administrationof CDDP followed by treatment with the CT20 peptide was the mosteffective in inducing cell death, which was not inhibited by ZVAD-FMK orBcl-2 (FIG. 6A). The expression of Bcl-2 by transfected cells wasconfirmed by immunoblotting lysates prepared from cells (FIG. 10).

In FIG. 10, cells were transiently transfected with Bcl-2, treated withnanoparticles/CT20. Lysates were immunoblotted for Bcl-2. Controls areuntransfected cells (UT) and untreated cells (control). * denotes anon-specific band. A representative blot of two performed is shown.

While Sytox is good indicator of cell death, it does not distinguishbetween apoptotic and necrotic cell death. Therefore, to determinewhether the CT20p peptide induced apoptotic cell death, the amount ofmembrane asymmetry caused by flipping of phospholipids phospholipids inthe plasma membrane using a violet ratiometric probe was determined. InFIG. 6B, dot blots show the comparison of loss of membrane integrity tochanges in membrane symmetry in MDA-MB-231 cells treated with CT20ppeptide and/or CDDP. Treatment with the CT20p peptide encapsulated innanoparticles enabled penetration of the DNA-binding dye (as shown inFIG. 6A), but did not promote changes in membrane symmetry. This wasminimally affected by caspase inhibition or Bcl-2 expression. Incontrast, contrast, CDDP induced significant alterations in the membranesymmetry detectable by the violet ratiometric probe, which alterationswere inhibited by ZVAD-FMK or Bcl-2 (FIG. 6B; data shown isrepresentative of more than three independent experiments). Thecombination treatment of CDDP and CT20p peptide was the most efficaciousand was minimally affected by caspase inhibition or Bcl-2 expression(FIG. 6B). These findings indicate that the CT20 peptide engages in adeath mechanism distinct from that of CDDP, and and that this distinctmechanism is independent of ZVD-FMK inhibited-caspases and is resistantto Bcl-2.

To demonstrate that the CT20 peptide could kill cancer cells in vivo, asmall scale murine tumor experiment was performed. MDA-MB-231 cells wereimplanted in the flanks of nude mice. Initial growth of tumors wasdetected after 2 weeks. At this time tumors were measured, and over a4-5 day period, the mice were treated with intratumoral injections ofPBS, unloaded or empty nanoparticles (NP), or CT20peptide/nanoparticles. The CT20 peptide prevented tumor growth ascompared to empty nanoparticles or a PBS-treated tumor. (FIG. 6C;results displayed in the graph show the change in tumor volume duringthe four days of treatment and are a representative of three independentexperiments). These results demonstrated that the CT20 peptide waseffective not only in tissue culture, but also in the tumor environment.

Moreover, the ultrasound data in FIG. 6D showed little to no observabledamage of normal tissue surrounding the regressing tumors treated withthe CT20 peptide. FIG. 6D provides representative ultrasound image ofchanges in tumor volume induced by treatment on days 0, 2 and 4 with theunloaded or CT20 peptide loaded nanoparticles. These findings indicatereduced uptake or effectiveness in non-cancerous cells.

Collectively, these data demonstrated that the C-terminal domain of Baxhas membrane-binding capacity. The CT20p peptide permeabilized membranesand caused cell death. The CT20 peptide, unlike the full-length Baxprotein, caused a lethal cascade that resulted in membrane rupture thatis not characteristic of conventional apoptosis. The amphipathicfeatures of the CT20 peptide made it amenable to encapsulation innanoparticles and delivery to cancer cells. The CT20 peptide caused thedeath of colon and breast cancer cells, even in the absence ofendogenous Bax or expression of Bcl-2, and led to reduced tumor volumein a murine model. The CT20 peptide caused cell death in a manner thatwas different from the apoptotic mechanism activated by CDDP.

Central to the lethal function of Bax is the membrane binding capacityof domains like the C-terminal alpha-9 helix. Loss of the C-terminus, ormutagenesis of K189/K190, modulated the ability of Bax to associate withmitochondria. The CT20 peptide, derived from the C-terminal a9 helix ofBax, penetrated and permeabilized lipid vesicles. The CT20 Bax peptide,when encapsulated in nanoparticles, was toxic at picomolarconcentrations. The CT20 Bax peptide/nanoparticle mixture inducedmorphological features of cell death such loss of membrane integrity andcell shrinkage, but did not cause the changes in membrane symmetry thatcharacterizes apoptosis. Use of the Bax CT20 peptide/nanoparticles as ananti-cancer agent was demonstrated when breast cancer cell linessuccumbed to treatment as indicated by increased loss of membraneintegrity that was not impaired by caspase inhibition or Bcl-2expression. The CT20 peptide caused a form of cell death that wasmechanistically different from typical anti-cancer drugs like CDDP.

The data indicate that the CT20p peptide kills cells caused theformation of pores in mitochondrial membranes. The CT20p peptide wasefficient at forming pores in zwitterionic and anionic lipid membranes,leading to the release of calcein from loaded lipid vesicles. FIG. 11,for example, shows a model for a membrane pore formed by the CT20 Baxpeptide. Control peptides did not permeabilize lipid membranes, causingcalcein release, which indicated that the observed pore formation wasspecific to the CT20 Bax peptide. Second-order rate kinetics revealedthat initial pore formation by the CT20p peptide was slow, which wasfollowed by a faster rate of assembly. The data indicate that the CT20ppeptide formed pores leading to membrane destabilization, ion exchange,and/or the release of sequestered molecules.

Pore-forming proteins or peptides that can spontaneously insert intolipid membranes and form stable pores have significant biologicalinterest and clinical application. As demonstrated herein, the CT20peptide, a shorter version of the α9 helix of Bax (amino acids 172-192),caused cell death indicated by membrane rupture. These findings indicatethat the CT20 peptide may cause necrotic-like cell death, rather thanconventional apoptosis, and therefore has the potential for additiveeffects in combinatorial therapies with agents like CDDP that induceapoptosis. The data presented herein show that a small amount of theCT20 peptide was lethal under conditions when the apoptotic machinery isintact (such as Bax-containing HCT-116 cells) or when the apoptosismachinery is defective (such as Bax deficient HCT-116 cells). The CT20peptide was also effective under conditions of effector caspaseinhibition or Bcl-2 over expression. These data indicate that the CT20peptide may be a potent killing tool in cancers with abnormal levels ofanti-apoptotic proteins, as well as cancers in which other survivalsignaling mechanism may be irregular. FIG. 12, for example, shows ahypothetical cell death pathway for CT20p peptide using a basic model ofapoptotic and non-apoptotic cell death.

Example 6. Pore Formation by CT20p Peptide

An analysis of formation of relatively large membrane pores by BaxC-terminal 20-reside peptide (CT20p) and two mutants where the twonative lysines are replaced either with glutamates (charge reversalmutation) or leucines (charge neutralization mutation) were examined.The three peptides demonstrate distinct potencies to form pores in bothzwitterionic and anionic membranes that transport calcein (M_(r)˜623).The most efficient pores are formed by the wild-type peptide in anionicmembranes. The kinetics of calcein release at various concentrations ofthe three peptides allowed identification of the second-order rateconstants of pore formation within the membrane, the affinity constantsof peptide units composing the pore, and the oligomeric pore structure.Nucleation of the pore is shown to be relatively slow and involve 2-3peptide molecules, followed by a faster process of assembly of the porethat includes up to eight peptide molecules. Structural studies led to amodel of an octameric transmembrane pore with an inner diameter of 20-22A. Analysis of the kinetics of calcein release from lipid vesiclesallows determination of rate constants of pore formation,peptide-peptide affinities within the membrane, the oligomeric state oftransmembrane pores, and the role of the lysine residues.

In summary, the C-terminal 20-residue stretch of Bax has the capabilityof forming relatively large membrane pores with a radius of at least 13Å. Replacement of the two lysine residues close to the C-terminus withanionic glutamate or nonpolar leucine residues reduce the pore formingactivity of the peptide, but the mutant peptides are still quite capablepore formers. In general, the pore formation is a two-stage process,nucleation and assembly of the final pore structure that includes up toeight peptide molecules, and is stabilized by intermolecular interactionenergies of −10 to −13 kcal/mol, which is quite significant for 20-merpeptides.

Additionally, the pore structure was analyzed by polarized Fouriertransform infrared, circular dichroism, and fluorescence experiments onthe peptides reconstituted in phospholipid membranes. The peptidesassumed an α/β-type secondary structure within membranes. Both β-strands and a-helices are significantly tilted relative to the membranenormal, by 30-60 degrees. The tryptophan residue embed into zwitterionicmembranes at 8-9 A from membrane center. Membrane anionic charge causesa deeper insertion of tryptophan for BaxC-KK and BaxC-LL but notBaxC-EE. Combined with pore stoichiometry, these data suggest a poremodel where eight peptide molecules form an “α/β-ring” structure withpore inner diameter of 20-22 Å. These results identified a strongmembranotropic activity of Bax C-terminus and proposed a new mechanismby which peptides can efficiently perforate cell membranes and thus beused as cytotoxic agents.

Additionally, membrane insertion of the peptide and subsequent poreformation was mediated mainly by hydrophobic rather than electrostaticinteractions. In the presence of bulk water, the peptides assume anα/β-type structure, with the β-strands and α-helices significantlytilted relative to the membrane normal. Altogether, the data areconsistent with an octameric “α/β-ring” structure with an internal porediameter of at least 13 Å to 20-22 Å that can effectively transfercalcein and larger molecules. The data establish a foundation forcharacterization of the molecular structure of the α/β ringtransmembrane pore formed by Bax-derived peptides.

Example of Killing Microbial Cells

FIG. 13 shows a comparison of CT20p peptde with antimicrobial peptidesand apoptosis-inducing peptides. Attributes are shared among thepeptides. FIG. 14 shows that Ct20p peptide inhibited the growth of E.coli, a gam negative bacteria, in a dose dependent manner, inhibiting50% of the growth of bacteria at a dose of 50 μg. The inhibition ofgrowth began around 3 hours and reached a plateau at 13 hours, seegraph. These results compared to control peptide XL or no peptide, inwhich growth began at 3 hours and plateaued at 6-7 hours. The E. coliwere grown in the presence of a peptide, concentrations of CT20p peptideor for control, SCR peptide, or no peptide for a control, in standardconditions for growing bacteria in broth media.

FIG. 15 shows counts of the bacterial colonies from the broth culture ofFIG. 14. The bacterial culture media was plated after 24 hours oftreatment/exposure to Ct20p or the control conditions, a peptide (SCR)or no peptide and addition of DMSO, and the resulting colonies werecounted. There were few bacterial colonies detected with CT20p treatmentof 24 hours, which indicated that the effects of CT20p peptide werebactericidal, not bacteriostatic (inhibition of growth). A conclusionwas that the inhibition of growth seen in FIG. 14 was due to the killingor lysing of bacteria.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. It will be apparent to those skilled in the art thatvarious modifications and variations can be made in the presentinvention without departing from the scope or spirit of the invention.

More specifically, certain agents which are both chemically andphysiologically related can be substituted for the agents describedherein while the same or similar results can be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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TABLE 1 F. Sequences Seq ID No. Description Sequence 1 CT20Bax WTVTIFVAGVLTASLTIWKKMG 2 CT20Bax EE VTIFVAGVLTASLTIWEEMG 3 CT20Bax LLVTIFVAGVLTASLTIWLLMG 4 CT20Bax RR VTIFVAGVLTASLTIWRRMG 5 BAX-KK (for)5′-GGATCACTCTCGGCCTGGACACCATGGG GATGTACCCATACGATGTTCCAGATTACGCTGACGGGTCCGGGGAGCAG-3′ 6 BAX-KK (rev)5′-CGTCGACTGCAGAATTCTCAGCCCATC TTCTTCCAGATGGTGAGCGAGG-3′ 7 BAX-ΔNT 5′-CCGGGGAGCAGCCCCATATGTACCCAT (1-19) (for) ACGATGTTCCAGATTACGCTATGAAGACAGGGGCCCTTTTGC-3′ 8 BAX-ΔNT  5′-CGTCGACTGCAGAATTCTCAGCCCATCTT(1-19) (rev) CTTCCAGATGGTGAGCGAGG-3′ 9 BAX-ΔCT 5′-GGATCACTCTCGGCCTGGACACCATGG (173-192)  GGATGTACCCATACGATGTTCCAG (for)ATTACGCTGACGGGTCCGGGGAGCAG-3′ 10 BAX-ΔCT 5′-CGTCGACTGCAGAATTCTCAGGTCTGCCA (173-192)  CGTGGGCGTCCAAAG-3′ (rev) 11BAX-LL (for) 5′-GGATCACTCTCGGCCTGGACACCATGG GGATGTACC CATACGA TGTTCCAGATTACGCTGACGGGTCCGGGGAGCAG-3′ 12 BAX-LL (rev)5′-CGTCGACTGCAGAATTCTCAGCCCATGA GGAGCCAGATGGTGAGCGAGG-3′ 13 BAX-DD (for)5′-GGATCACTCTCGGCCTGGACACCATG GGGATGTACCCATACGATGTTCCAGATTACGCTGACGGGTCCGGGGAGCAG-3′ 14 BAX-DD (rev) 5′-CGTCGACTGCAGAATTCTCAGCCCATGTCGTCCCAGATGGTGAGCGAGG-3′ 15 BAX-EE (for)5′-GGATCACTCTCGGCCTGGACACCATGGG GATGTACCCATACGATGTTCCAGATTACGCTGACGGGTCCGGGGAGCAG-3′ 16 BAX-EE (rev)5′-CGTCGACTGCAGAATTCTCAGCCC ATCTCCTCCCAGATGGTGAGCGAGG-3′ 17 BAX-RR (for)5′-GGATCACTCTCGGCCTGGACACCATG GGGATGTACCCATACGATGTTCCAGATTACGCTGACGGGTCCGGGGAGCAG-3′ 18 BAX-RR (rev)5′-CGTCGATCTCAGCCCATTCGTCGCC ACATGGTGAGCGAGG-3′ 19 BAX-QQ (for)5′-GGATCACTCTCGGCCTGGACACCAT GGGGATGTACCCATACGATGTTCCAGATTACGCTGACGGGTCCGGGGAGCAG-3′ 20 BAX-QQ (rev)5′-CGTCGACTGCAGAATTC TCAGCCCATC TGCTGCCAGATGGTGAGCGAGG-3′ 21BAX-KMGK (for) 5′-GGATCACTCTCGGCCTGGACACCATG GGGATGTACCCATACGATGTTCCAGATTACGCTGACGGGTCCGGGGAGCAG-3′ 22 BAX-KMGK (rev)5′-CGTCGACTGCAGAATTCTCACTTCCCCA TCTTCCAGATGGTG AGCGAGG-3′ 23BAX-EK (for) 5′-GGATCACTCTCGGCCTGGACACCATG GGGATGTACCCATACGATGTTCCAGATTACGCTGACGGGTCCGGGGAGCAG-3′ 24 BAX-EK (rev)5′-CGTCGACTGCAGAATTCTCAGCCCA TCTTCTCCCAGATGGTGAGCGAGG-3′ 25 GFP-CT-WT 5′-GGATCACTCTCGGCCTGGACGA (for) GGATATCATGGTGAGCAAG-3′ 26 GFP-CT-WT 5′-CGTCGACTGCAGATATCTCAGCCCA (rev) TCTTCTTCCAGATGGTGAGCGAGGCGGTGAGCACTCCCGCCACAAAGATG GTCACGGTGTTATCTAGATC-3′ 27 GFP-CT-EE 5′-GGATCACTCTCGGCCTGGACGA (for) GGATATCATGGTGAGCAAG-3′ 28 GFP-CT-EE 5′-CGTCGACTGCAGATATCTCAGCCCATC (rev) TCCTCCCAGATGGTGAGCGAGGCGGTGAGCACTCCCGCCACAAAGATGG TCACGGTGTTATCTAGATC-3′ 29 GFP-CT-RR 5′-GGATCACTCTCGGCCTGGACGA (for) GGATATCATGGTGAGCAAG-3′ 30 GFP-CT-RR 5′-CGTCGACTGCAGATATCTCAGCCCATG (rev) AGGAGCCAGATGGTGAGCGAGGCGGTGAGCACTCCCGCCACAAAGATGG TCACGGTGTTATCTAGATC-3′ 31 DD-CT-WT (for)5′-AATTCTGTGACCATCTTTGTGGCGGGA GTGCTCACCGCCTCGCTCACCATCTGGAAGAAGATGGGCTGA-3′ 32 DD-CT-WT (rev) 5′-GATCTCAGCCCATCTTCTTCCAGATGGTGAGCGAGGCGGTGAGCACTCCCG CCACAAAGATGGTCACAG-3′ 33 DD-CT-EE (for)5′-AATTCTGTGACCATCTTTGTGGC GGGAGTGCTCACCGCCTCGCTCACCATCTGGGAGGAGATGGGCTGA-3′ 34 DD-CT-EE (rev) 5′-GATCTCAGCCCATCTCCTCCCAGATGGTGAGCGAGGCGGTGAGCA CTCCCGCCACAAAGATGGTCACAG-3′ 35 DD-CT-LL (for)5′-AATTCTGTGACCATCTTTGTGGCGGGA GTGCTCACCGCCTCGCTCACCATCTGGCTCCTCATGGGCTGA-3′ 36 DD-CT-LL (rev) 5′-GATCTCAGCCCATGAGGAGCCAGATGGTGAGCGAGGCGGTGA GCACTCCCGCCACAAA GATGGTCACAG-3′ 37 DD-CT-RR (for)5′-AATTCTGTGACCATCTTTGTGGCGGGA GTGCTCACCGCCTCGCTCACCATCTGGCGACGAATGGGCTGA-3′ 38 DD-CT-RR (rev) 5-GATCTCAGCCCATTCGTCGCCAGATGGTGAGCGAGGCGGTGAGCAC TCCCGCCACAAA GATGGTCACAG-3′

What is claimed is:
 1. A method for treating cancer in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a composition comprising a C-terminal B celllymphoma-2 (Bcl-2)-associated X protein (Bax) peptide (CT20p peptide)and a pharmaceutically acceptable carrier, wherein the CT20p peptideconsists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, or SEQ ID NO: 4; and wherein the cancer is selected from thegroup consisting of breast cancer, colorectal cancer, lung cancer, andliver cancer.
 2. The method of claim 1, further comprising administeringto the subject an anti-cancer drug.
 3. The method of claim 2, furthercomprising administering to the subject a chemotherapeutic drug.
 4. Themethod of claim 2, further comprising administering to the subject aradiosensitizer.
 5. The method of claim 1, wherein the CT20p peptide isadministered directly into the cancer.
 6. The method of claim 1, furthercomprising repeating the administration of the therapeutically effectiveamount of the composition comprising the CT20p peptide and thepharmaceutically acceptable carrier.
 7. The method of claim 1, whereinthe CT20p peptide is encapsulated in a nanoparticle.
 8. The method ofclaim 7, wherein the nanoparticle is aminated.
 9. The method of claim 7,wherein the nanoparticle is carboxylated.
 10. The method of claim 7,wherein the nanoparticle is a polymeric nanoparticle.
 11. The method ofclaim 10, wherein the polymeric nanoparticle is a hyperbranchedpolymeric (HBPE) nanoparticle.